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MYCOTAXON 


THE INTERNATIONAL JOURNAL OF FUNGAL TAXONOMY & NOMENCLATURE 


VOLUME 120 APRIL-JUNE 2012 


Manoharachariella indica sp. nov. 
(Rajeshkumar & Singh pp. 43-48— Conidia) 
KUNHIRAMAN C, RAJESHKUMAR, artist 


ISSN (PRINT) 0093-4666 http://dx.doi.org/10.5248/120 ISSN (ONLINE) 2154-8889 
MYXNAE 120: 1-510 (2012) 


EDITORIAL ADVISORY BOARD 


HENNING KNUDSEN (2008-2013), Chair 
Copenhagen, Denmark 


SEPPO HUHTINEN (2006-2012), Past Chair 
Turku, Finland 


WEN-YING ZHUANG (2003-2014) 
Beijing, China 

ScoTT A. REDHEAD (2010-2015) 
Ottawa, Ontario, Canada 

SABINE HUHNDORE (2011-2016) 
Chicago, Illinois, U.S.A. 

PETER BUCHANAN (2011-2017) 
Auckland, New Zealand 


Published by 
MycoTaxon, LTD. 
P.O. BOX 264, ITHACA, NY 14581-0264, USA 


www.mycotaxon.com & www.ingentaconnect.com/content/mtax/mt 
© Mycotaxon, LTp, 2012 


MYCOTAXON 


THE INTERNATIONAL JOURNAL OF FUNGAL TAXONOMY & NOMENCLATURE 


VOLUME 120 


APRIL-JUNE, 2012 


EDITOR-IN-CHIEF 


LORELEI L. NORVELL 


editor@mycotaxon.com 
Pacific Northwest Mycology Service 
6720 NW Skyline Boulevard 
Portland, Oregon 97229-1309 USA 


NOMENCLATURE EDITOR 


SHAUN R. PENNYCOOK 


PennycookS@LandcareResearch.co.nz 
Manaaki Whenua Landcare Research 
Auckland, New Zealand 


BooK REVIEW EDITOR 


ELSE C. VELLINGA 


bookreviews@mycotaxon.com 
861 Keeler Avenue 
Berkeley CA 94708 U.S.A. 


CONSISTING OF I-XII + 510 PAGES INCLUDING FIGURES 


ISSN 0093-4666 (PRINT) http://dx.doi.org/10.5248/120.cvr ISSN 2154-8889 (ONLINE) 


© 2012. MycoTAxon, LTD. 


IV ... MYCOTAXON 120 


MY COTAXON 


VOLUME ONE HUNDRED TWENTY — TABLE OF CONTENTS 


COVER SECTION 


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REVICWENE. 6 in Gas ge 8 nS 8M gh Maced bole a LOSS BY SS Ghee Make eh ated OP RM Ags ix 
SUDHMUSSION PrOCCUUTCS ss Bee 5 255A yt oR Aer Ae RAs wh td eho ape ees x 
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RESEARCH ARTICLES 
Glomus trufemii (Glomeromycetes), a new sporocarpic species 
from Brazilian sand dunes Bruno Tomio Goto, Jomar Gomes Jardim, 
Gladstone Alves da Silva, Eduardo Furrazola, Yamir Torres-Arias & Fritz Oehl 
Two new species of Pluteus section Celluloderma from the 
Dominican Republic Alfredo Justo, Eliseo Battistin & Claudio Angelini 
The lichen genus Chapsa (Graphidaceae) in India 
Santosh Joshi, Dalip K. Upreti & Sanjeeva Nayaka 
A new species of Inonotus (Hymenochaetaceae) and Trametes cingulata 
(Polyporaceae) newly recorded from Brazil 
Maira Cortellini Abrahao & Adriana de Mello Gugliotta 
Manoharachariella indica sp. nov. from the Western Ghats, India 
Kunhiraman C. Rajeshkumar & Sanjay K. Singh 
Russula jilinensis sp. nov. from northeast China 
Guo-Jie Li, Sai-Fei Li, Xing-Zhong Liu & Hua-An Wen 
Acrogenospora hainanensis sp. nov. and new records of microfungi 
from southern China 
Jian Ma, Li-Guo Ma, Yi-Dong Zhang, Ji-Wen Xia & Xiu-Guo Zhang 
A new species of Nectria on Populus from China 
Zhao-Qing Zeng & Wen-Ying Zhuang 
A new species of the lichen genus Phlyctis from Maharashtra, India 
Gayatri Chitale & Urmila Makhija 
Two new freshwater species of Annulatascaceae from China 
Dian-Ming Hu, Lei Cai, Ali Hassan Bahkali & Kevin D. Hyde 
New records of little-known species of Carbomyces 
(Carbomycetaceae, Ascomycota) Gabriel Moreno, Marcos Lizarraga, 
Martin Esqueda, Ricardo Galan & Pablo Alvarado 
Cantharellus zangii, a new subalpine basidiomycete from 
southwestern China 
Xiao-Fei Tian, Bart Buyck, Shi-Cheng Shao, Pei-Gui Liu & Yan Fang 
Seven lichen species new to Poland 


11 


89 


99. 


Martin Kukwa, Anna Lubek, Rafat Szymczyk & Anna Zalewska 105 


APRIL-JUNE 2012... V 


Dissoconium proteae newly recorded from China Rong Zhang, 
Yongna Mao, Lu Hao, Hongcai Chen, Guangyu Sun & Mark L. Gleason 119 
The fungal collection of the Jaiellonian University Herbarium (KRA), 
Krakow, Poland Anna Maria Ociepa, Szymon Zubek & Piotr Mleczko 127 
Mycena pseudoinclinata, new to Italy 
Alfonso La Rosa, Alessandro Saitta, Riccardo Compagno, & Giuseppe Venturella133 
Rossbeevera yunnanensis (Boletaceae, Boletales), a new 
sequestrate species from southern China 
Takamichi Orihara, Matthew E. Smith, Zai-Wei Ge & Nitaro Maekawa 139 
Octaviania violascens: a new sequestrate bolete 
from Thailand Rattaket Choeyklin, Thitiya Boonpratuang, 
Sujinda Sommai & Sayanh Somrithipol 149 
New records of cercosporoid hyphomycetes from Iran 
Mahdi Pirnia, Rasoul Zare, Hamid R. Zamanizadeh & Akbar Khodaparast 157 
Phaeocollybia nigripes (Agaricomycetes), a new species from Brazil 
Victor R.M. Coimbra, Tatiana B. Gibertoni & Felipe Wartchow 171 
Peziza michelii and its ectomycorrhizae with Alnus nitida 
(Betulaceae) from Pakistan T. Ashraf, M. Hanif & A.N. Khalid 181 
Erysiphe javanica sp. nov., a new tropical powdery mildew 
from Indonesia Jamjan Meeboon, Iman Hidayat & Susumu Takamatsu 189 
Pisolithus: A new species from southeast Asia and a new combination 
C. Phosri, M.P. Martin, N. Suwannasai, P. Sihanonth & R. Watling 195 
Terriera simplex, a new species of Rhytismatales from China 
Xiao-Ming Gao, Chun-Tao Zheng & Ying-Ren Lin 209 
The Leptogium juressianum complex in southeastern Brazil 
Marcos J. Kitaura & Marcelo P. Marcelli 215 
Coriolopsis psila comb. nov. (Agaricomycetes) and two new 
Coriolopsis records for Brazil Georgea S. Nogueira—Melo, 
Priscila S. de Medeiros, Allyne C. Gomes-Silva, Leif Ryvarden, Helen M.P. Sotao 
& Tatiana B. Gibertoni 223 
A new species of Postia (Basidiomycota) from Northeast China 
Bao-Kai Cui & Hai-Jiao Li 231 
Three new Caeoma species on Rosa spp. from Pakistan 
N.S. Afshan, A.N. Khalid & A.R. Niazi 239 
Nomenclatural status and morphological notes on 
Tubifera applanata sp. nov. (Myxomycetes) D.V. Leontyev & K.A. Fefelov 247 
Paralepistopsis gen. nov. and Paralepista 


(Basidiomycota, Agaricales) Alfredo Vizzini & Enrico Ercole 253 
Three new species of Septobasidium (Septobasidiaceae) from Hainan Province 
in China Suzhen Chen & Lin Guo 269 


Characterization of the causal agent of poplar anthracnose occurring 
in the Beijing region Zheng Li, Cheng Ming Tian & Ying Mei Liang 277 


VI... MYCOTAXON 120 


Sympodioplanus yunnanensis, a new aquatic species from 
submerged decaying leaves Guang-Zhu Yang, Kai-Ping Lu, Yue Yang, 
Li-Bo Ma, Min Qiao, KeQing Zhang & Ze-Fen Yu 

Phaeotrichoconis crotalariae, endophytic on Vitis labrusca 
Thais Emanuelle Feijé de Lima, José Luiz Bezerra 
& Maria Auxiliadora de Queiroz Cavalcanti 


287 


291 


Studies on Wrightoporia from China 1. A new species from Hunan Province, 


South China Jia-jia Chen & Hai-You Yu 
A new species of Bipolaris from Iran Abdollah Ahmadpour, 
Zeinab Heidarian, Maryam Donyadoost-Chelan, 
Mohammad Javan-Nikkhah & Takao Tsukiboshi 
Notes on Xylophallus xylogenus (Phallaceae, Agaricomycetes) 
based on Brazilian specimens 
Larissa Trierveiler-Pereira & Rosa Mara Borges da Silveira 
Plectania seaveri sp. nov. (Ascomycota, Pezizales), a new discomycete 
from Bermuda Matteo Carbone, Carlo Agnello & Scott LaGreca 
New species of Entoloma (Basidiomycetes, Agaricales) from 
Kerala State, India C.K. Pradeep, K.B. Vrinda, Shibu P. Varghese & TJ. Baroni 
A new species of Infundichalara from pine litter Ondiej Koukol 
Clavulicium hallenbergii, a new corticioid species from India 
Avneet P. Singh, Jaspreet Kaur & G.S. Dhingra 
Vararia longicystidiata sp. nov. (Agaricomycetes) from India 
Samita, S.K. Sanyal, G.S. Dhingra & Avneet P. Singh 
A new cystidiate variety of Omphalina pyxidata (Basidiomycota, 
Tricholomatoid clade) from Italy Alfredo Vizzini, 
Mariano Curti, Marco Contu & Enrico Ercole 
Four new records of Aspergillus sect. Usti 
from Shandong Province, China Long Wang 
Systematics of the Gomphales: the genus Gomphus sensu stricto 
Admir J. Giachini, Carla M. Camelini, 
Marcio J. Rossi, Claudio R.ES. Soares & James M. Trappe 
Myxomycetes from China 15: Arcyria galericulata sp. nov. 
Bo Zhang, Tian-Hao Li, Qi Wang & Yu Li 
New records of Puccinia species on Poaceae from Pakistan 
N.S. Afshan, A.N. Khalid & A.R. Niazi 
Lectotypification and characterization of the natural phenotype of 
Fusarium bactridioides Keith A. Seifert & Tom Grafenhan 
Lectotypification of Crepidotus variabilis var. subsphaerosporus 


295 


301 


309 


S17 


331 
343 


353 


357 


361 


373 


385 


401 


407 


415 


Sona Jancovicova & Shaun R. Pennycook 423 


A new species of Conidiobolus (Ancylistaceae) from Anhui, China 
Yong Nie, Cui-Zhu Yu, Xiao-Yong Liu & Bo Huang 


427 


APRIL-JUNE 2012... 


First record of the sequestrate fungus Neosecotium macrosporum 
(Agaricales, Lepiotaceae) for Mexico Marcos Lizarraga, 
Martin Esqueda, Mario Vargas-Luna & Gabriel Moreno 
New combinations in Lactifluus. 3. L. subgenera Lactifluus and Piperati 
A. Verbeken, K. Van de Putte & E. De Crop 
Exobasidium ferrugineae sp. nov., associated with hypertrophied flowers 
of Lyonia ferruginea in the southeastern USA 
Aaron H. Kennedy, Nisse A. Goldberg & Andrew M. Minnis 
Passalora aseptata, a new cercosporoid fungus from 
northeastern Uttar Pradesh, India Raghvendra Singh, 
Balmukund Chaurasia, Kalawati Shukla & Parmatma Prasad Upadhyaya 
First records of Craterium aureonucleatum, Perichaena quadrata, 
and Physarum mutabile in Italy Riccardo Compagno, 
Alfonso La Rosa, & Giuseppe Venturella 
Tuber microsphaerosporum and Paradoxa sinensis spp. nov. 
Li Fan, Jin-Zhong Cao & Yu Li 
Entomophthoromycota: a new phylum and reclassification 


for entomophthoroid fungi Richard A. Humber 
Moniliophthora aurantiaca sp. nov., a Polynesian species 
occurring in littoral forests Bradley R. Kropp & Steven Albee-Scott 


ABSTRACTS OF NEW MYCOBIOTAS ONLINE 
Checklist of the Argentinean Agaricales 2. 
Coprinaceae and Strophariaceae N. Niveiro & E. Alberté 
Checklist of the Argentinean Agaricales 3. 
Bolbitiaceae and Crepidotaceae N. Niveiro & E. Alberté 
Preliminary checklist of the macromycetes from Collestrada 
forest ecosystems in Perugia (Italy) Paola Angelini, Giancarlo Bistocchi, 
Andrea Arcangeli & Roberto Venanzoni 
New, rare, and noteworthy lichens in the Pollino National Park 
(Basilicata, southern Italy) 
D. Puntillo, M. Puntillo, G. Potenza & S. Fascetti 
Saprobic fungi on wood an litter of Alnus alnobetula in the Swiss Alps 
Beatrice Senn-Irlet, Rolf Miirner, Elia Martini, Nicolas Kiiffer, 
Romano de Marchi & Guido Bieriice 


NOMENCLATURE 
Nomenclatural novelties proposed in volume 120 


VII 


437 


443 


451 


461 


465 


471 


477 


493 


505 


505 


505 


506 


506 


507 


vu ... MYCOTAXON 120 


ERRATA FROM PREVIOUS VOLUMES 


VOLUME 119 


p. 97, lines 37-38 for: unpigmented lower two cells. read: pigmented basal cell. 


p. 411, line 15 for: Korea Forest Institute read: Korea Forest Research Institute 


PUBLICATION DATE FOR VOLUME ONE HUNDRED NINETEEN 
MYCOTAXON for JANUARY-MARCH, VOLUME 119 (I-x1I + 1-511) 
was issued on April 11, 2012 


APRIL-JUNE 2012... IX 


REVIEWERS — VOLUME ONE HUNDRED TWENTY 


The Editors express their appreciation to the following individuals who 
have, prior to acceptance for publication, reviewed one or more of the 
papers prepared for this volume. 


Reinhard Agerer 

M. Catherine Aime 

Joe Ammirati 

Vladimir Antonin 

Takayuki Aoki 

André Aptroot 

Mohammad Bahram 

Richard Baird 

Hans-Otto Baral 

Timothy J. Baroni 

Reinhard Berndt 

Franco Umbertomaria 
Bersan 

Janusz Blaszkowski 

Wolfgang von Brackel 

Uwe Braun 

Peter Buchanan 

Lori M. Carris 

Rafael FE. Castafieda Ruiz 

Michael A. Castellano 

Efren Cazares Gonzalez 

Pedro Crous 

Yu-Cheng Dai 

Kanad Das 

Cvetomir M. Denchev 

Stephanos Diamandis 

Maria Martha Dios 

Pradeep K. Divakar 

Uno Eliasson 

John A. Elix 

Francisco C.O. Freire 

Walter Gams 

Laura Guzman-Davalos 

Nils Hallenberg 

Shuanghui He 

Rosanne Healy 

José R. Hernandez 

M. Luciana 
Hernandez Caffot 


Adriana Inés Hladki 
Kentaro Hosaka 
Peter Jeffries 
Mikael Jeppson 
Alfredo Justo 
Gintaras Kantvilas 
Taiga Kasuya 
Bryce Kendrick 
Paul M. Kirk 
Roland Kirschner 
Henning Knudsen 
Richard P. Korf 
Hanns Kreisel 
Carlos Lado 
Teresa Lebel 
Bernardo E. Lechner 
Xiao- Yong Liu 
Guozhong Lit 
Quan Li 
Robert Licking 
Eric H.C. McKenzie 
Alistair McTaggart 
Nelson Menolli, Jr. 
Andrew M. Minnis 
David W. Minter 
David W. Mitchell 
Pierre-Arthur Moreau 
Gabriel Moreno 
Jurga Motiejunaite 
Edna Dora M. 
Newman Luz 
Lorelei L. Norvell 
Eduardo R. Nouhra 
Clark L. Ovrebo 
Giovanni Pacioni 
Omar Paino Perdomo 
Ka-Lai Pang 
Shaun R. Pennycook 
Ronald H. Petersen 


Stephen W. Peterson 
Alan J.L. Phillips 
Marcin Piatek 
Donald H. Pfister 
Huzefa Raja 
Scott A. Redhead 
Gerardo Lucio Robledo 
Andrea Irene Romero 
Amy Y. Rossman 
James Scott 
Mark R.D. Seaward 
Keith A. Seifert 
Hyeon-Dong Shin 
Ewald Sieverding 
Rosa Mara Bourges da 
Silveira 
Takuya Sakoda 
B.M. Sharma 
Hyeon-Dong Shin 
Joost A. Stalpers 
Brett Summerell 
Guangyu Sun 
Michal TomSovsky 
Mauro Tretiach 
Ricardo Valenzuela 
Nicolas Van Vooren 
Carlos G. Vélez 
Else. C. Vellinga 
Annemieke Verbeken 
Rytas Vilgalys 
Yu-Lian Wei 
Anthony J. S. Whalley 
Zhu-Liang Yang 
Ming Ye 
Hai-Sheng Yuan 
Xiu-Guo Zhang 
Zhongyi Zhang 


x ... MYCOTAXON 120 


FOUR EASY STEPS TO SUCCESSFUL MYCOTAXON PUBLICATION IN 2012 


Prospective Mycoraxon authors should download instructions PDF, review and 
submission forms, and other helpful templates by clicking the ‘file download page’ link 
on our INSTRUCTIONS TO AUTHORS page before preparing their manuscript. Below is a 
summary of our “4-step’ publication process. 


1—PEER REVIEW: Email formatted text and illustration files with a2012 MycoTaxon 
Reviewer Comments Form to 2-3 experts for peer review. Authors should (i) ask 
peer reviewers to return revisions and comment forms to BOTH authors and Editor- 
in-Chief <editor@mycotaxon.com> and (ii) revise the files according to reviewer 
suggestions before sending text files to the Nomenclature Editor for nomenclatural 
review. 


2—NOMENCLATURAL REVIEW: Email text files (wiTH tables & captions but No 
artwork) to the Nomenclature Editor <PennycookS@LandcareResearch.co.nz> 
for accession and pre-submission review. The Email message MUST include 
‘Mycotaxon’ on the subject line AND all peer reviewer names+Email addresses in 
the message. The Nomenclature Editor will assign accession numbers and return 
annotated files with a list of needed corrections to the authors and Editor-in-Chief. 


3—FINAL SUBMISSION: After again consulting experts and revising manuscripts as 
needed, send the (i) completed 2012 MycoTaxon submission form; (ii) separate 
text files for main text, tables, and legends; and (iii) art files to the Editor-in-Chief 
<editor@mycotaxon.com>. Only text and image files prepared for immediate 
publication should be sent at this time. The Editor-in-Chief usually acknowledges 
manuscripts and thanks expert reviewers within two weeks, but please wait at 
least 14 days before sending a follow-up query (without attachments); this helps 
us keep Email traffic to a minimum during Mycotaxon publication deadlines or 
temporary closures of the editorial office. 


4—FINAL EDITORIAL REVIEW & PRESS PREPARATION: Files not ready for publication 
will be rejected or returned to authors for further revision; the Editor-in-Chief gives 
tentatvely approved manuscripts a final grammatical and scientific review before 
converting all files into publishable format. The PDF proof, bibliographic citation, 
and nomenclatural entries are sent to all coauthors for final inspection prior to 
publication. After PDF conversion, the Editor-in-Chief corrects only processing or 
editorial errors prior to publication, but corrections of author errors are listed in 
the Errata of a subsequent volume for no charge. If authors have selected the open 
access option, they are asked to arrange payment of page charges with the Business 
Manager <subscriptions@mycotaxon.com> at this time. 


MyYcoTAXxON LTD— www.mycotaxon.com 

The Mycotaxon Webmaster <mycotaxon@gmail.com> posts general and 
subscription information, important announcements, and author forms and templates 
on the official MycoTaxon site, which also hosts the regional mycobiota webpage for 
free download of distributional annotated species lists. 


MYCOTAXON ONLINE— www.ingentaconnect.com/content/mtax/mt 
Mycotaxon publishes four 500-page volumes a year. Both open access and 
subscription articles are offered. 


APRIL-JUNE 2012... XI 


FROM THE EDITORS 


MYCOTAXON, DELAYS, AND 2012 — In 1973 Dick Korf and Gregoire Hennebert noted 
that taxonomic papers were unnecessarily delayed by traditional blind peer review and 
press processing. They developed Mycotaxon to eliminate these frustrating bottlenecks 
by having authors select their own expert reviewers (thus directing the review pace) 
and prepare print ready copy. This innovative procedure was enthusiastically received. 
Papers were published quickly, usually within three months after submission, and 
Mycotaxon established an enviable reputation for reliability and efficiency. Complaints, 
if any, focused on the ‘unprofessional’ appearance of the journal. Begun in the days 
of typewritten copy, the journal permitted each author to select their own style and 
formatting, resulting in a somewhat ‘haphazard’ appearance for each volume. 


Nine years ago, our Editorial Advisory Committee suggested that ‘MycoTaxon 
style’ move from oxymoron to reality. With authors more or less computer literate, the 
new Editor-in-Chief could require electronic submission and expand the ‘Instructions 
to Authors’ to set forth the new style and formatting requirements. Happily, by early 
2005, all submissions arrived on the editorial desk via Email, except for an occasional 
illustration-heavy CD sent by regular post. 

Adding our Nomenclature Editor in 2005 ensured thoroughly vetted nomenclature; 
with two editors revising each manuscript, fewer errors were published and most papers 
were published 3 to 6 months after final submission. Our impact factor rose steadily 
through 2011. 

We still strive to process manuscripts and publish as speedily as possible. Unfortunately 
our speed and efficiency rest on a manageable number of manuscripts (~250 annually) 
& emails— and Shaun's and my health. 2012 has brought a ‘perfect storm’ for delays 
— manuscripts pouring in at an accelerated rate and unexpected medical ‘issues’ for both 
Editors. The early April publication of MycoTaxon 119 was followed by eye surgery and 
major orthopedic surgery for Shaun, worrying authors accustomed to nomenclatural 
reviews returned within 1-2 weeks (needlessly so, however, as all manuscripts are still 
processed in the order received). Shaun fortunately is now back at the nomenclatural desk 
and hard at work on the 70+ manuscripts that accumulated during his recuperations. 

Understandably, my treatment of the cancer diagnosed in July Has delayed this 
volume by three months, as I can no longer put in the 70-80 hours per week on Email 
and revising, reformatting, and processing manuscripts that I have since 2004. With my 
final reviews for MycoTAxoNn 121 only now beginning, the July-September volume will 
also be late. Ever optimistic, however, I still hope that Mycotaxon 122 will be delayed 
only one month, (not three!). [I dream! ] 

We take this opportunity to thank our authors for their patience and understanding. 
We feel that MycoTaxon remains the most comprehensive journal for fungal taxonomy 
and nomenclature. It should remain so for years to come and should arrive once more in 
a predictably timely fashion after resuming its normal schedule in 2013. 

Warm regards, 

LorELEI NoRVELL (Editor-in-Chief) with 
SHAUN PENNYCOOK (Nomenclature Editor) 
22 September 2012 


ISSN (print) 0093-4666 © 2012. Mycotaxon, Ltd. ISSN (online) 2154-8889 


MY COTAXON 


http://dx.doi.org/10.5248/120.1 
Volume 120, pp. 1-9 April-June 2012 


Glomus trufemii (Glomeromycetes), a new sporocarpic species 
from Brazilian sand dunes 


BRUNO TOMIO GOTO”, JOMAR GOMES JARDIM', GLADSTONE ALVES DA SILVA’, 
EDUARDO FURRAZOLA?, YAMIR TORRES-ARIAS? & FRITZ OEHL‘ 


"Departamento de Botanica, Ecologia e Zoologia, CB, Universidade Federal do 
Rio Grande do Norte, Campus Universitario, 59072-970, Natal, RN, Brazil 
*Departamento de Micologia, CCB, Universidade Federal de Pernambuco, 
Av. Prof. Nelson Chaves, S/N, CEP 50670-420, Cidade Universitaria, Recife, PE, Brazil 
*Instituto de Ecologia e Sistemdatica, IES-CITMA, A.P. 11900, La Habana, Cuba 
‘Federal Research Institute Agroscope Reckenholz-Tanikon ART, Organic Farming Systems, 
Reckenholzstrasse 191, CH-8046 Ziirich, Switzerland 


‘CORRESPONDENCE TO: brunogoto@hotmail.com 


ABSTRACT — Glomus trufemii, which forms large aggregates (< 850 x 1200 um) in the 
rhizosphere of a herbaceous plant community in NE Brazilian sand dunes, is described as 
new. Its subglobose glomerospores measuring 72-92 x 79-105 um have two spore wall layers: 
an evanescent hyaline 0.3-0.8 um thick outer layer and a laminate orange brown to dark red 
brown 7.4—15.5 um thick inner layer. The pigmentation of the subtending hypha is similar but 
often much lighter than that of the laminate spore wall layer. The spore size and color, spore 
wall structure, and features of the spore base and subtending hyphae separate this species 
from similar yellow-brown to brown spored species like G. badium, G. glomerulatum, and 
G. brohultii. 


Key worps — morphology, Glomeromycota, Glomerales, restinga 


Introduction 

The Tulasne brothers described the genus Glomus in 1844 for G. macrocarpum 
(the type species according to Clements & Shear 1931). Since then many other 
fungi with glomoid spores like those of G. macrocarpum have been named, 
so that now Glomus comprises >60 species. For many years Glomus was 
considered monophyletic based solely on morphological evidence. Morton 
& Redecker (2001), based on morphological and molecular data, erected the 
genera Paraglomus (Paraglomeraceae) and Archaeospora (Archaeosporaceae), 
whose members also formed glomoid spores. Schwarzott et al. (2001) inferred 
from phylogenetic SSU rDNA molecular analyses that Glomus is non- 


2 ... Goto & al. 


monophyletic. Schiifler et al. (2001) subsequently erected a new phylum, 
Glomeromycota, in which Glomus was represented by three phylogenetically 
different groups: A, B, and C. Oehl & Sieverding (2004) transferred glomoid 
species with two spore walls (with the inner wall serving as a germinal wall) 
to their new genus Pacispora. Thereafter Walker & Schiifler (2004) transferred 
Glomus spurcum C.M. Pfeiff. et al. to the newly established genus Diversispora 
and order Diversisporales. 

To date, glomoid glomerospores can be found in species representing 
Archaeosporales, Diversisporales, Glomerales, and Paraglomerales, making 
their identification difficult in the absence of molecular data. Morphological 
identification has been facilitated recently in a thorough review of glomoid 
spore-forming species (Oehl et al. 2011), which introduced helpful suggestions 
based on molecular data associated with morphological differences among the 
major phylogenetic clades in the Glomeromycota. Our description of G. trufemii 
is based exclusively on the new key morphological criteria, and the species fits 
the Oehl’s et al. (2011) revised concept of Glomus. 


Material & methods 


Study area and sites 

Samples were collected in the Parque Estadual das Dunas de Natal “Jornalista Luiz Maria 
Alves,’ the first Conservation Unit created in the Municipality of Natal, Rio Grande do 
Norte State and currently one of the largest urban conservation areas with dune vegetation 
in Brazil. The site is located at 5°46'S 35°12’W with soils analyzed as pH (H,O) = 
5.5-5.75, P = 2-4 mg kg", 2,86 g dm? of organic matter. The climate is tropical rainy 
(type Am of K6éppen) with a short four-month dry period, with a 25.5 °C mean annual 
temperature and 1191 mm mean annual precipitation. In the sand dune ecosystem, the 
typical ‘restinga vegetation varies from herbaceous to shrubs and trees (Oliveira-Filho 
1993, Oliveira-Filho & Carvalho 1993). The park is composed of mainly represented 
by tree species of the Anacardiaceae, Bignoniaceae, Fabaceae, Myrtaceae, and Rubiaceae 
(Anacardium occidentale, Caesalpinia echinata, Campomanesia dichotoma, Chamaecrista 
ensiformis, Eugenia ligustrina, Guettarda platypoda, Myrcia guianensis, Myrciaria 
tenella, Tabebuia roseoalba, Tocoyena sellowiana) and herbaceous species of Poaceae, 
Cyperaceae, Asteraceae, Araceae, and Rubiaceae. 


Morphological analyses 

Glomerospores and sporocarps were separated from the soil samples by sucrose 
gradient in plate dishes (Blaszkowski et al. 2006). Sporocarps and glomerospores were 
placed in dishes with water and separated under a dissecting microscope. Sporocarps were 
fragmented to study spore organization. Glomerospores were mounted on microscope 
slides either in water (to check unmodified characteristics of spore wall components and 
spore colour; Spain 1990) or permanently in PVLG (Omar et al. 1979) or PVLG with 
Melzer's reagent (Koske & Tessier 1983). 

Terminology follows Oehletal. (2011) and Furrazolaetal. (2011). Thespore denomination 
of Goto & Maia (2006) was used. Zeiss Axioskop compound microscopes with or without 


Glomus trufemii sp. nov. (Brazil) ... 3 


Nomarski differential interference contrast (DIC) were used for detailed spore observations; 
digital images were taken with either an Axiocam camera and AxioVision (v. 3.1 software at 
1300 x 1030 dpi) or Canon digital cameras. 


Arbuscular mycorrhizal cultures 

Bait cultures for the soils from sand dunes (Parque das Dunas de Natal) with spores 
and sporocarps were established in the greenhouse of the Universidade Federal do Rio 
Grande do Norte with Zea mays L. as host plant, using sterilized soil and 0.5 L pots. 

Pure cultures of the new fungus were attempted by inoculating Zea mays seedlings 
germinated in autoclaved quartz sand with G. trufemii sporocarps isolated from sand 
dunes. After filling 0.5 L pots with sterilized sand dune soil, we formed a small hole in 
the center using a glass shaker and discharged spores and sporocarps into the hole using 
Pasteur pipettes. Then a Z. mays seedling was placed into the hole, covered with sand, 
and grown for one cycle of 3-4 months. The fungus did not produce spores in either bait 
cultures grown even for 9-12 months or pure cultures. 


Taxonomy 


Glomus trufemii B.T. Goto, G.A. Silva & Oehl, sp. nov. FIGs. 1-9 
MycoBank MB 561567 


Differs from G. brohultii by more regularly shaped subtending hyphae that are pigmented 
further below the spore bases and darker colored orange to red brown spores with 
slightly thicker walls. 


Type: Brazil, Rio Grande do Norte, near Natal, Parque das Dunas de “Natal”, sand dunes 
with herbaceous plant community on a convex hillside, 15.Feb.2010, B.T. Goto, in 
permanent slide (PVLG) (Holotype: UFRN1482; Isotype: UFRN1483, ZT Myc 15118). 


EryMo_oey: trufemii = in honor of Sandra Farto Botelho Trufem, taxonomist, who 

long taught many young students and researchers in arbuscular mycorrhizal fungal 

taxonomy. 
SPOROCARPS formed in aggregates (500-850 x 780-1200 um), orange brown to 
dark red brown adherent to living or dead roots with hundreds to thousands of 
glomerospores; rarely singly in soils. Melzer’s reaction not observed. Peridium 
absent. Sporocarpic hyphae hyaline (3.8—)7.6-10.4(-12.8) um without a septum 
and with interwoven arrangements. 

GLOMEROSPORES formed terminally on hyphae, subglobose to elliptical 
(72-92 x 79-105 um) or rarely globose (72-96 um diam.), orange brown when 
young to dark red brown at maturity, perhaps darkening slightly to dark brown 
when ageing in soil. 

SPORE WALL 7.4-15.5 um thick overall, consisting of two layers (swtl, 
SWL2); SwWL1 thin (0.3-0.8 um), hyaline and difficult to observe in mature 
spore, but generally visible in the subtending hyphal wall; sw12 thick (7.4-15.5 
um), pigmented orange brown to dark red brown, laminated, smooth. Spore 
wall layers continuous with subtending hyphal wall layers. The pigmentation of 
SWL2 continues into the subtending hyphal wall, but this layer is often lighter 


4 ... Goto &al. 


Hyphae 


™ Septum 


yy Aborted 
100um 


* sw 


*® swi1 


Glomus trufemii sp. nov. (Brazil) ... 5 


in color in the subtending hypha. swil and sw12 do not stain when exposed 
to Melzer’s reagent. 

SUBTENDING HYPHA (SH) generally present, single, straight or constricted, 
cylindrical to sharply curved, light yellow to dark yellow; 5.1-12.7 um diam. 
(mean-8.8 tm) at the spore base, the hyphal wall 2.6-5.1 um thick (mean 
4.2 um) near the spore base and tapering to approx. 1 um at 15 um from the 
base; occlusion formed by introverted sw12 thickening and often through 
an additional bridging septum arising from sw1L2, but sometimes the pore 
appearing partly open. 

ABORTED GLOMEROSPORES frequently observed and forming terminally on 
hyphae, generally collapsed or subglobose to elliptical (35-56 x 40-46 um), light 
yellow to dark yellow, possibly darkening slightly to light brown when aging in 
soils. Spore wall layer (swL2) (<3.8 um) not staining in Melzer’s reagent. 

GERMINATION structures unknown. 

GLOMEROSPORE DEVELOPMENT was deduced from positively identified 
spores in aggregates found in different developmental stages. The hyaline hyphal 
wall layer differentiates into a hyaline, evanescent spore wall layer (swL1) and 
then a laminate layer (swL2) that becomes more pigmented with increasing 
numbers of developing sublaminae. After the glomerospores mature, their pore 
is closed by introverted thickening of sw12 and an additional bridging septum 
arising from the laminate wall layer. 

ARBUSCULAR MYCORRHIZA FORMATION remains unknown. 

DISTRIBUTION — So far, the new fungus was detected only in Brazil. 
Known in soil from sand dunes mainly harbouring herbaceous species of 
Araceae, Asteraceae, Cyperaceae, Poaceae and Rubiaceae (e.g.: Acroceras 
zizanioides (Kunth) Dandy, Anthurium affine Schott, Aspilia procumbens 
Baker, Centratherum punctatum Cass., Cyperus maritimus Poir., C. meyenianus 
Kunth, Mitracarpus eichleri K.Schum., Raddia brasiliensis Bertol., Richardia 
grandiflora (Cham. & Schltdl.) Steud.) growing in native, ecologically unstable 
restinga ecosystems in Parque das Dunas, Natal (Brazil). 


Discussion 

Glomus species are very common in sand dunes (Blaszkowski 2010; 
Blaszkowski et al. 2009a,b, 2010a,b) and several new species have been 
described recently (G. africanum Blaszk. & Kovacs, G. achrum Blaszk. et al., 


Fics. 1-9. Glomus trufemii. 1. Sporocarps in PVLG; note the numerous glomerospores in this 
part of sporocarp. 2-3. Fragment of sporocarp with healthy glomerospores and aborted spores. 
4, Aborted spores with only one spore wall layer (sw12). 5. Septum formed by sw12. 6. Spores 
in different developmental stages. 7. Spore wall layers in mature spore (SwL1&2). 8-9. Spore wall 
layers (SwL1&2) continuous with subtending hyphal wall (SW) that form distinct pigmentation. 


6 ... Goto & al. 


G. bistratum Btaszk. et al., G. indicum Blaszk. et al., G. iranicum Blaszk. et al., 
G. majewskii Btaszk., G. perpusillum Blaszk. & Kovacs). Glomus trufemii is 
readily distinguished from previously described sporocarpic Glomus species 
by glomerospore size and colour, spore wall structure, (including that at the 
spore base), and characters of the subtending hypha. Most of these species form 
colourless or pale-coloured spores singly or in loose aggregates (Blaszkowski 
et al. 2009a,b; 2010a,b) and thus are easy to distinguish from the sporocarpic 
dark-spored G. trufemii. Sporocarps of G. trufemii slightly resemble spore 
ageregates of G. aggregatum N.C. Schenck & G.S. Sm. and G. fasciculatum 
(Thaxt.) Gerd. & Trappe (Schenck & Smith 1982, Gerdemann & Trappe 1974). 
However, the G. fasciculatum sw12 stains in Melzer’s reagent (vs. no reaction 
in G. trufemii). Spores of G. aggregatum generally are irregular in shape (vs. 
globose to subglobose) and have much thinner spore and subtending hyphal 
walls. The spore wall of G. trufemii is one of the thickest among the known 
glomeromycotan species with glomoid spores. 

Several Glomus spp. form sporocarps without a peridium with similarly 
sized and coloured spores similarly distributed in a sporocarp: G. ambisporum 
G.S. Sm. & N.C. Schenck, G. atrouva McGee & Pattinson, G. badium Oehl et 
al., G boreale (Thaxt.) Trappe & Gerd., G. botryoides RM. Rothwell & Victor, 
G. brohultii Sieverd. & R.A. Herrera, G. flavisporum (M. Lange & E.M. Lund) 
Trappe & Gerd., G. glomerulatum Sieverd., G. heterosporum G.S. Sm. & N.C. 
Schenck, G. invermaium LR. Hall, and G. macrocarpum Tul. & C. Tul. (Oehl et 
al. 2011). However, of these only G. badium, G. brohultii, and G. glomerulatum 
also form spores with subtending hyphae generally clearly lighter-coloured 
than the spore wall (Sieverding 1987, Herrera-Peraza et al. 2003, Oehl et al. 
2005, Oehl et al. 2011). In the other species, the wall color of both spore and 
subtending hyphae is similar or identical (Oehl et al. 2011). 

Glomus glomerulatum produces intercalary yellow brown glomerospores 
having 2-4 subtending hyphae (vs only terminal spores with one subtending 
hypha in G. trufemii). Moreover, G. trufemii spores are darker (orange-brown 
to dark reddish brown) and have a significantly thicker wall. 

Glomus badium forms irregularly sized 3-layered spores around an 
intrasporocarpic gleba, while G. trufemii generally produces regularly sized 
subglobose 2-layered spores in sporocarps lacking glebas. 

Glomus trufemii and G. brohultii spores have a similar size and wall structure. 
Also, although G. brohultii was not originally described as forming sporocarps 


Fics. 10-18. Glomus brohultii. Bi-layered spores (swL1&2). 10. Glomerospores in PVLG; note the 
subtending hypha (SH) which is pigmented up to ca. 50 um below the spore base before becoming 
subhyaline to hyaline. 11-12. Spores frequently have two subtending hyphae. 13. Mature spore with 
sw12 thickened at the spore base. 14. Irregular subtending hypha becoming hyaline in a rather 
short distance from the spore base (20-30 um). 16. Hyaline subtending hypha with two layers 
(SHL1&2). 17-18. Crushed spores with two spore wall layers (swL1&2). 


Glomus trufemii sp. nov. (Brazil) ... 7 


8 ... Goto & al. 


or aggregates, sporocarps have been observed recently (Oehl, pers. obs.) during 
AMF diversity studies in cultivated white and yellow yam sites and adjacent 
natural savannas in West Africa (Tchabi et al. 2009), ruling out sporocarp 
or aggregate formation as a diagnostic character separating G. trufemii and 
G. brohultii. However, G. brohultii spores are paler and lack orange or red 
ones— yellow-brown to brown, not orange-brown to dark red-brown — and 
its spore walls are generally thinner. Furthermore, in G. trufemii the subtending 
hyphal pigmentation extends further below the spore base, both in spores 
formed in sporocarps or singly in the soil. Finally, the shape of the subtending 
hyphae is much more irregular in G. brohultii (Herrera-Peraza et al. 2003: this 
paper, Fics. 10-18). 


Acknowledgements 

The authors acknowledge, in special, Dr. Janusz Blaszkowski (Department of 
Plant Protection, West Pomeranian University of Technology, Szczecin, Poland) and 
Dr. Ewald Sieverding (Institute for Plant Production and Agroecology in the Tropics 
and Subtropics, University of Hohenheim), for reviewing the manuscript and making 
helpful comments and suggestions and appreciate the corrections by Shaun Pennycook, 
Nomenclatural Editor, and suggestions by Lorelei L. Norvell, Editor-in-Chief. The study 
was supported by the Universidade Federal de Pernambuco which provided a grant to 
FE Oehlas ‘visiting professor and Programa de Pés-Graduac¢ao em Sistematica e Evolugao 
that invited F. Oehl for a technical visit to Natal, RN. 


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ISSN (print) 0093-4666 © 2012. Mycotaxon, Ltd. ISSN (online) 2154-8889 


MY COTAXON 


http://dx.doi.org/10.5248/120.11 
Volume 120, pp. 11-21 April-June 2012 


Two new species of Pluteus section Celluloderma 
from the Dominican Republic 


ALFREDO JUSTO”, ELISEO BATTISTIN* & CLAUDIO ANGELINI? 


'Clark University. Biology Department, 950 Main St., Worcester 01610 MA USA 
?Natural History Museum, Corso Italia 63, Valdagno 36078 Italy 

°Via Tulipifero 9, Porcia 33080 Italy 

* CORRESPONDENCE TO: ajusto@clarku.edu 


ABSTRACT— Two new species of Pluteus sect. Celluloderma collected in the Dominican 
Republic are described based on morphological and molecular (nrITS) characters. Pluteus 
crenulatus is characterized by the sulcate crenulate margin of the pileus, clavate cheilo- and 
caulocystidia, and absence of pleurocystidia. Pluteus stenotrichus is characterized by the 
presence of very narrow and internally septate cells in the pileipellis. 


Key worps— biodiversity, Caribbean, phylogeny, Pluteaceae, taxonomy 


Introduction 

The genus Pluteus Fr. (Pluteaceae, Agaricales) has been the focus of recent 
molecular phylogenetic studies (Menolli et al. 2010; Justo et al. 2011a,b), 
which essentially support the traditional subdivision of the genus in three 
sections (Pluteus, Celluloderma Fayod, and Hispidoderma Fayod) but with 
some rearrangements. Sect. Celluloderma still accommodates all species with 
non-metuloid hymenial cystidia and a pileipellis arranged as an hymeniderm 
or epithelium composed mostly of clavate or spheropedunculate elements 
(intermixed or not with elongated cells) but now, based on molecular data, also 
includes taxa with a cutis-type pileipellis (previously assigned to sect. Villosi 
Schreurs & Vellinga or Hispidoderma) or with distinct partial veil (previously 
assigned to Chamaeota (W.G. Sm.) Earle) (Menolli et al. 2010; Justo et al. 
2011a,b; Vizzini & Ercole 2011). 

Here we describe two new Pluteus species recently collected in the Dominican 
Republic. Morphological and molecular (nrITS) data support their inclusion 
in sect. Celluloderma. Both taxa are compared with morphologically similar 
species and their phylogenetic position is discussed. 


12 ... Justo, Battistin & Angelini 


Materials & methods 

Collections were studied using standard procedures for morphological examination of 
Pluteus (e.g., Justo & Castro 2007, Minnis & Sundberg 2010). Descriptive morphological 
terms follow Vellinga (1988). The notation [30, 2, 1] indicates that measurements were 
made of 30 basidiospores from 2 basidiocarps in 1 collection. Color codes are from 
Munsell Soil-Color Charts (Munsell Color 2009). The following abbreviations are used 
in the descriptions: avl for average length, avw for average width, Q for the length/width 
quotient and avQ for average quotient. 

Standard procedures for DNA isolation, PCR, and sequencing were applied (e.g., 
Justo et al. 2011la,b). The “nuclear ribosomal internal transcribed spacers 1 and 2 
including the 5.8S region” is abbreviated as nrITS. The final dataset includes sect. 
Celluloderma nrITS sequences generated by Justo et al. (2011b), additional sequences 
generated by the first author, and sequences currently available in GenBank (mostly 
originated by O’Brien et al. 2005, Matheny et al. 2006, Midgley et al. 2007, Malysheva et 
al. 2009, Menolli et al. 2010). All accession numbers and sequence geographic origins 
are given in Fic. 1. Sequences were aligned using MAFFT version 6 (http://mafft.cbre. 
jp/alignment/server/; Katoh & Toh 2008) with the Q-INS-i option, and alignments 
were manually corrected using MacClade 4.05 (Maddison & Maddison 2002) before 
deposition in TreeBASE (http://purl.org/phylo/treebase/phylows/study/TB2:S12007). 
A Maximum Likelihood analysis was run using the RAxML servers with 100 rapid 
bootstrap replicates (http://phylobench.vital-it.ch/raxml-bb/index.php; Stamatakis et 
al. 2008). Pluteus cervinus (Schaeff.) P. Kumm. and P. petasatus (Fr.) Gillet (both sect. 
Pluteus) were used as outgroup taxa. 


Results 

The phylogenetic position of the new taxa described here is highlighted 
in Fic. 1 and discussed in detail under Taxonomy. Sequence identifications 
marked with “***” (which require further attention but fall outside the scope 
of this paper) are listed here to avoid causing further taxonomic confusion 
in GenBank: (i) “Pluteus cf. nanus” (HM562046) and “P. nanus” (jF908611) 
probably represent P. cinereofuscus J.E. Lange; (ii) “Pluteus cinereofuscus” 
(JF908616) is part of the Pluteus nanus (Pers.) P. Kumm. species complex; (iii) 
“Pluteus cervinus” (JF908623) represents a taxon close to Pluteus ephebeus 
(Fr.) Gillet; (iv) “Pluteus galeroides”( JF908610, JF908609) probably represents 
Pluteus chrysophlebius (Berk. & M.A. Curtis) Sacc.; (v) “Pluteus boudieri” 
(jF908627) represents Pluteus phlebophorus (Ditmar) P. Kumm.; (vi) Pluteus 
insidiosus (JF908626) falls within collections morphologically assignable to 
Pluteus thomsonii (Berk. & Broome) Dennis. 


FIGURE 1. Best tree from the Maximum Likelihood analysis of Pluteus sect. Celluloderma. Bootstrap 
values 270% are shown on or below the branches. Root length has been reduced to facilitate 
graphical representation. 


Pluteus spp. nov. (Dominican Republic) ... 13 


HQ654907 Pluteus aurantiorugosus var aurantiovelatus Italy 


i HQ654908 Pluteus aurantiorugosus var aurantiovelatus Italy 
New taxa decribed JF908608 Pluteus aurantiorugosus Italy 
1 1 A HM562041 Pluteus aurantiorugosus Spain ° 
In this article JF908613 Pluteus aurantiorugosus Italy Pluteus aurantiorugosus 


100 | HM562121 Pluteus aurantiorugosus Japan 
AT ate od, 4 HM562081 Pluteus aurantiorugosus Illinois 
%& Mixtini type pileipellis HM562074 Pluteus aurantiorugosus Michigan 
72L HM562072 Pluteus aurantiorugosus Massachusetts 
9] 98 JF908630 Pluteus romellii Italy 


indi HM562062 Plut Ilii_ Spai 
Pleurocystidia absent [> [sie Acs nei san 
FJ774073 Plut llii Russi ip 
or very scarce 75 HM562123 splutsdesronielit Jonah Pluteus romellii 
100 HM562078 Pluteus romellii Michigan] 


87], HM562183 Pluteus romellii Illinois 
964 HM562105 Pluteus romellii Missouri 
79,- HM562160 Pluteus aureovenatus Brazil 
FJ816663 Pluteus aureovenatus Brazil 
100 _}_ Aygs4065 Pluteus aff romellii California Pluteus aureovenatus complex 
FJ816667 Pluteus sublaevigatus Brazil 
100 HM562173 Pluteus sp. Brazil —————_ —__— Pluteus sp.O 
JN603201 Pluteus stenotrichus AJ352 Dominican Republic * 
HMS562161 Pluteus sp. Brazil ————————_—— P]  teus Sp. 
HM562198 Pluteus aff ephebeus England 
100] HM562080 Pluteus aff ephebeus France 
88] | JF908623 Pluteus cervinus Italy*** 
199, JF908620 Pluteus ephebeus Italy 
76 JF908621 Pluteus ephebeus Italy Pluteus ephebeus complex 
HM562044 Pluteus ephebeus Spain 
HM562162 Pluteus riberaltensis var conquistensis Brazil 
100 HM562091 Pluteus fenzlii Japan ma 
100] FJ774082 Pluteus fenzlii Russia Pluteus fenzlii 
100 HM562111 Pluteus fenzlii Slovakia 
HM562086 Pluteus mammillatus Missouri al 
HM562119 Pluteus mammillatus Missouri Pluteus mammillatus 
100L M562120 Pluteus mammillatus Florida 


98, HM562122 Pluteus podospileus Japan | s 
100 HM562049 Pluteus podospileus Spain Pluteus podospileus * 


83 HM562196 Pluteus aff podospileus Sweden| Pluteus aff. podospileus * 


HM562199 Pluteus seticeps Illinois 


100__| HM562192 Pluteus seticeps wisconsin Pluteus seticeps *0O 


HM562191 Pluteus seticeps Missouri 
100 HM562076 Pluteus eliae Florida H 
Nc00008 Plates ae mii | Pluteus eliae / Pluteus sp. 
76, FJ816657 Pluteus jamaicensis Brazil : . 
100] § FJ816662 Pluteus fuligineovenosus Brazil Pluteus fluminensis com plex 
ap FJ816664 Pluteus fluminensis Brazil 
FJ816655 Pluteus fluminensis Brazil 


100 , HM562146 Pluteus sp. | Pluteus sp. 


HM562148 Pluteus sp. Brazil 
HM562145 Pluteus sp. Brazil | Pluteus Sp. 
HM562046 Pluteus cf. nanus Spain *** 
JF908611 Pluteus nanus Italy *** ° 
B2 HM562108 Pluteus cinereofuscus Portugal Pluteus cinereofuscus 
HM562124 Pluteus cinereofuscus Spain 
FJ774085 Pluteus eludens Russia 
HM562118 Pluteus eludens Madeira Island Pluteus eludens * 
76) 9°L HM562185 Pluteus eludens Illinois 
HM562056 Pluteus pallescens Spain | Pluteus pallescens 
81 HM562115 Pluteus aff cinereofuscus Japan 


87L AY969369 Uncultured basidiomycete North eet Pluteus aff. cinereofuscus 


HM562201 Pluteus multiformis Spain ° . 
88 DQ672275 Uncultured soil basidiomycete austrata| Pluteus multiformis %& /Pluteus Sp. 

HM562138 Pluteus phlebophorus Spain 

72] HM562137 Pluteus phlebophorus Spain 
HM562039 Pluteus phlebophorus Spain 

21 JF908627 Pluteus boudieri Italy*** 
HMS562144 Pluteus phlebophorus Spain Pluteus phlebophorus 
HM562184 Pluteus phlebophorus Michigan 

87. a HM562112 Pluteus phlebophorus Michigan 
“| HEHM562193 Pluteus pallidus Michigan 


75 HM562117 Pluteus phlebophorus Japan 
0.09 HM562186 Pluteus aff phlebophorus Illinoist Pluteus aff. phlebophorus 


HM562116 Pluteus cf eugraptus Japan] Pluteus cf. eugraptus * 


JF908616 Pluteus cinereofuscus Italy*** 
JF908633 Pluteus nanus Italy 
_ £4774081 Pluteus nanus Russia Pluteus nanus com plex 
HM562216 Pluteus sp VI Illinois 
JN603203 Pluteus cf nanus AJ348 Spain 
96 80, JF908610 Pluteus galeroides Italy*** 
75 91/1 jF908609 Pluteus galeroides Italy*** 
HM562064 Pluteus chrysophlebius Spain “ 
96 4M562088 Pluteus cheepeniesits seo Pluteus chrysophlebius 
74] 'HM562125 Pluteus chrysophlebius Japan 
HM562181 Pluteus chrysophlebius Illinois 
81) 1 Hms62182 Pluteus chrysophlebius Illinois 
64 (90 HM562180 Pluteus chrysophlebius Illinois oe 
HM562079 Pluteus rugosidiscus Michiganl Pluteus rugosidiscus 
100) HM562066 Pluteus thomsonii Spain 
fa0 JF908626 Pluteus insidiosus Italy*** 
HM562067 Pluteus thomsonii Illinois oe 
| )£1774082 Pluteus thomsonii Rusia | Pluteus thomsonii complex %O 
100} HMS562053 Pluteus thomsonii Spain 
100 JF908607 Pluteus thomsonii Italy 
HM562197 Pluteus thomsonii Michigan 


FJ816665 Pluteus dominicanus var hyalinus Brazil Pluteus dominicanus var. hyalinus 


JN603202 Pluteus crenulatus AJ353 Dominican Republic 


HM562143 Pluteus dietrichii Spain 'Pluteus dietrichiiO 


HM562038 Pluteus petasatus Spain 


100 HM562133 Pluteus cervinus Spain | Outgroup (sect. Pluteus) 


96 


14 ... Justo, Battistin & Angelini 
Taxonomy 


Pluteus crenulatus Justo, Battistin & Angelini, sp. nov. FIG. 2 
MycoBAank 563469 


Similar to Pluteus tucumanus, also with a sulcate pileus with crenulate margin, 
but differing in broader spores, clavate cheilo- and caulocystidia, and absence of 
pleurocystidia 


Type: Dominican Republic, Prov. Puerto Plata, Sosua, Union, 17 Jan 2011, leg. C. 
Angelini, coll. AJ353 (Holotype MICH; GenBank nrITS JN603202. Isotype MCVE 
27198). 


EryMo_oey: crenulatus refers to the distinctive crenulate pileus margin. 


PiLEus 20-40 mm, convex to plano-convex, with a broad umbo, deeply sulcate 
in the outer half; surface smooth or minutely granulose at center, granulose 
towards the margin; brown, darker at center [7.5YR 3/4, 4/6] and paler towards 
margin [7.5YR 5/6, 5/8, 6/6] where it alternates brown and whitish zones, not 
hygrophanous, margin distinctly crenulate, white. LAMELLAE crowded, free, 
ventricose; < 4mm broad; white when young, later pink with flocculose whitish 
edges. STIPE 30-50 x 2-4 mm, cylindrical, slightly broadened at base; white or 
white-cream; smooth or pruinose. CONTEXT in stipe and pileus white. SMELL 
indistinct. TASTE not recorded. SPORE PRINT not recorded, probably pink. 

BASIDIOSPORES [30, 2, 1] 4.5-5.5(-6.0) x 3.9-5.0(-5.5) um, avl x avw = 4.9 x 
4.2 um, Q = 1.00-1.26, avQ = 1.17, globose to broadly ellipsoid. BAsip1a 20-35 
x 9-12 um, 4-spored, clavate or narrowly utriform. PLEUROCYSTIDIA absent. 
LAMELLAR EDGE Sterile, completely covered with cystidia. CHEILOCYSTIDIA 
24-73 x 12-34 um, (narrowly) clavate, a few narrowly utriform; colorless; 
with thin, smooth walls; crowded, forming a well-developed strip. PILEIPELLIS 
a hymeniderm with transitions towards and epithelium; individual elements 
29-61 x 14-49 um, mostly clavate or spheropedunculate, a few narrowly 
utriform, mucronate, or with basal septum; filled with brown intracellular 
pigment, evenly dissolved or more rarely with pigment condensations; with 
thin, smooth walls. STrPITIPELLIs a cutis; hyphae 4-13 um wide, cylindrical, 
colorless or with brown pigment; with thin, smooth walls. CAULOCYSTIDIA 
38-51 x 17-31 um, (narrowly) clavate, some multiseptate; hyaline or with 
brown intracellular pigment, some with additional incrusting pigment; with 
thin to slightly thickened (less than 1 um), smooth walls; all over stipe surface 
in tightly packed clusters. CLAMP CONNECTIONS absent in all tissues. 

HABITAT AND DISTRIBUTION—Gregarious, on a fallen log in broad-leaved 
forest, January. Known only from the type locality in the Dominican Republic. 


ComMENtTS—The deeply sulcate pileus with a crenulate margin is the 
most distinctive macroscopic feature of P crenulatus. Microscopically, it is 
characterized by the clavate cheilo- and caulocystidia (Fic. 2) and the absence 
of pleurocystidia. 


Pluteus spp. nov. (Dominican Republic) ... 15 


FIGURE 2. Pluteus crenulatus (holotype AJ353). a. Basidiocarps; b. Basidiospores; c. Cheilocystidia; 
d. Pileipellis elements; e. Caulocystidia. Scale bars = 10 um. 


16 ... Justo, Battistin & Angelini 


P. tucumanus is a South American species that also has a sulcate, 
appendiculate-crenate pileus (Singer & Digilio 1952; Singer 1956, 1958). The 
following observations are based on our examination of its isotype in MICH 
(Singer T921 Argentina, Prov. Tucuman, Rio de los Sosas, 18 Jan 1950): 


Pluteus tucumanus Singer, Lilloa 25: 269. 1952 [“1951”]. Fic. 3 

BASIDIOSPORES [30, 1, 1] 3.9-5.0 x 2.7-4.0 um, avl x avw = 4.5 x 3.6 um, 
Q = 1.00-1.34(-1.69), avQ = 1.26, globose to ellipsoid, very rarely oblong. 
BasIDIA 25-31 x 7-10 um, 4-spored, narrowly utriform. PLEUROCYSTIDIA 
40-53 x 18-22 um, (narrowly) utriform with a distinct pedicel; colorless; 
with thin, smooth walls; scarce and scattered, present all over lamellar sides. 
LAMELLAR EDGE sterile, completely covered with cystidia. CHEILOCYSTIDIA 
29-52 x 12-32 um, (narrowly) utriform or broadly fusiform, a few narrowly 
clavate; colorless; with thin, smooth walls; crowded, forming a well-developed 
strip. Pileipellis an hymeniderm with transitions towards and epithelium; 
individual elements 38-49 x 21-32 um, mostly clavate or spheropedunculate; 
filled with brown intracellular pigment, evenly dissolved or more rarely with 
pigment condensations; with thin, smooth walls. STIPITIPELLIS a cutis; hyphae 
5-13 um wide, cylindrical, colorless or with brown pigment; with thin, smooth 
walls. CauLocystTip1a 38-51 x 17-31 um, (narrowly) clavate to (narrowly) 
utriform; hyaline or with brown intracellular pigment; with thin, smooth walls; 
all over stipe surface, scattered or in clusters. CLAMP-—CONNECTIONS absent in 
all tissues. 

Although the differences between P crenulatus and P. tucumanus are subtle 
(the latter distinguished by narrower spores, presence of pleurocystidia, and 
differently shaped cheilo- and caulocystidia), such subtle variations have proved 
critical in separating species in sect. Celluloderma (Justo et al. 2011c). 

Singer (1958) placed Pluteus fallax Singer and P iguazuensis Singer into 
the same morphological group (stirps Tucumanus) as P. tucumanus. We were 
unable to examine either species, but based on Singer's (1958) descriptions both 
taxa differ from P crenulatus in (among other characters) the non-crenulate 
pileus margin and presence of pleurocystidia. Singer also emphasized the 
presence of pigment condensations in the pileipellis elements of P. tucumanus 
and the absence of such condensations in P fallax and P. iguazuensis. We 
observed pigment condensations in both P crenulatus and P. tucumanus, but 
the taxonomic significance of this feature needs further study. 

Macroscopically, P crenulatus does not resemble any species in sect. 
Celluloderma with very rare to absent pleurocystidia — e.g., P diettrichii 
Bres., P. insidiosus Vellinga & Schreurs, P. poliocnemis Kihner — which also 
differ microscopically in spore and cystidia shape and size (see Kthner & 
Romagnesi (1956) and Vellinga (1990) for full descriptions). Pluteus thomsonii 


Pluteus spp. nov. (Dominican Republic) ... 17 


O09 (Vac 


FIGURE 3. Pluteus tucumanus (isotype, Singer T921). a. Basidiospores; b. Pleurocystidia; 
c. Cheilocystidia; d. Pileipellis elements; e. Caulocystidia. Scale bars = 10 um. 


and P. seticeps (G.F. Atk.) Singer, which sometimes have very rare or absent 
pleurocystidia, differ from P crenulatus by the “Mixtini-type’ pileipellis 
(see Minnis & Sundberg (2010) and Vellinga (1990) for full descriptions). 

Phylogenetically, P crenulatus appears somewhat isolated as sister to all taxa 
in sect. Celluloderma except P. diettrichii Bres. (Fic. 1). This placement receives 
no statistical support but suggests that P. crenulatus is not closely related to any 
of the major lineages presently recognized in sect. Celluloderma (Justo et al. 
2011b). 


Pluteus stenotrichus Justo, Battistin & Angelini, sp. nov. Fic. 4 
MycoBAnk 563470 

Characterized by a “Mixtini-type” pileipellis with very narrow, internally septate 
cylindrical elements. 
Type: Dominican Republic, Prov. Puerto Plata, Sosua, El Castillo, 16 Jan 2011, leg. C. 
Angelini, coll. AJ352 (Holotype MICH; GenBank nrITS JN603201. Isotype MCVE 
27197). 
ETYMOLOGy: stenotrichus (= “with narrow hairs”) refers to the very unusual type of 
pileipellis cells. 


18 ... Justo, Battistin & Angelini 


PILEuS (20-)25-50(-60) mm, convex to plano-convex, with or without a low 
broad umbo; surface distinctly rugose-venose at and around center, radially 
areolate-rimose in the outer half, exposing the white context; brown, darker at 
center [7.5YR 2.5/2, 2.5/3, 3/2, 3/3], paler around center [7.5YR 4/6, 5/6, 5/8] 
and much paler at margin [7.5YR 7/4, 7/6, 7/8], not hygrophanous; margin 
rimose but not striate or sulcate. LAMELLAE crowded, free, ventricose; < 7 mm 
broad; white or cream when young, later pink [5YR 8/3, 8/4], with concolorous 
or paler edges. StrPE 30-60 x 4-6 mm, cylindrical, slightly broadened at base; 
white or white-cream; smooth or slightly pruinose. CONTEXT in stipe and 
pileus white. SMELL indistinct. TASTE not recorded. SPORE PRINT not recorded, 
probably pink. 

BASIDIOSPORES [30, 2, 1] 4.9-7.0 x 4.5-5.7 um, avl x avw = 5.6 x 5.1 um, 
Q = 1.00-1.18, avQ = 1.11, globose to broadly ellipsoid. Bastp1a 25-34 x 
7.5-10 um, 4-spored, clavate or narrowly utriform. PLEUROCYSTIDIA 34-83 
x 14-32 um, (narrowly) utriform or lageniform, a few broadly conical with 
long pedicel; colorless; with thin, smooth walls; relatively common, scattered 
over lamellar sides. LAMELLAR EDGE sterile, completely covered with 
cystidia. CHEILOCYSTIDIA 25-50(-65) x 14-20(-25) um, (narrowly) clavate, 
spheropedunculate, some broadly fusiform, some with basal septum; colorless; 
with thin, smooth walls; crowded, forming a well-developed strip. PILEIPELLIS 
a hymeniderm with transitions towards and epithelium, composed of three 
types of elements: (i) globose, clavate or spheropedunculate 31-76 x 22-60 
um; (ii) narrowly clavate or lageniform 53-88 x 15-29 um; (iii) vey narrow 
elongated elements, 60-123 x 7-10(-12) um, cylindrical or flexuous, with 0-4 
internal septa, usually arising from a basal element < 15 um wide; all elements 
filled with brown intracellular pigment, more rarely hyaline; with thin, smooth 
walls. STIPITIPELLIS a cutis; hyphae 5-15 um wide, cylindrical, colorless or with 
brown pigment; with thin, smooth walls. CauLocystip1a 31-63 x 12-24 um, 
(narrowly) clavate, narrowly utriform or conical with basal septum; hyaline or 
with brown intracellular pigment; with thin, smooth walls; scattered all over 
stipe surface, isolated or in loosely arranged clusters. CLAMP CONNECTIONS 
absent in all tissues. 

HABITAT AND DISTRIBUTION—Gregarious, apparently terrestrial (among 
woody remnants and organic matter) in broad-leaved forest, January. Known 
only from the type locality in the Dominican Republic. 


COoMMENTS—The very narrow and internally septate elements in the 
pileipellis (Fic. 4) are diagnostic for P stenotrichus. Taxa with both clavate- 
spheropedunculate and elongated elements in the pileipellis have been 
traditionally classified in subsect. Mixtini Singer, but molecular data suggest 
that elongated elements in the pileipellis have evolved several times within 
sect. Celluloderma, rendering subsect. Mixtini artificial (Justo et al. 2011a, 


Pluteus spp. nov. (Dominican Republic) ... 19 


FiGURE 4. Pluteus stenotrichus (holotype AJ352). a. Basidiocarps; b. Basidiospores; 
c. Pleurocystidia; d. Cheilocystidia; e. Caulocystidia; f. Pileipellis elements - fl clavate 
and spheropedunculate, f2 narrowly clavate and lageniform, f3 very narrow and internally 
septate. Scale bars = 10 um. 


20 ... Justo, Battistin & Angelini 


Fig. 1). An extensive literature search and comparison with all species described 
with a “Mixtini-type” pileipellis (e.g. Horak 1964, 2008; Horak & Heinemann 
1978; Minnis & Sundberg 2010; Orton 1986; Pegler 1977, 1986; Singer 1956, 
1958; Vellinga 1990; see also Justo et al. 2011a,b,c for additional taxonomic 
references) found none with this type of septate elongated element. 

Pluteus stenotrichus is not closely related to any previously sequenced 
species with a “Mixtini-type” pileipellis (Fic. 1) and its position in the 
romellii/aurantiorugosus group is rather unexpected based on morphology. 
Pluteus aurantiorugosus (Trog) Sacc., P. romellii (Britzelm.) Sacc., and taxa 
in the species complex around P. aureovenatus Menolli & Capelari all lack 
any elongated pileipellis elements and are characterized in part by the bright 
yellow or orange-red colors of the pileus and/or stipe. The closest relative of 
P. stenotrichus is an undescribed species from Brazil (nrITS HM562173, 
collection $P394383) but that taxon differs in the (among other characters) 
absence of elongated pileipellis elements and almost total absence of hymenial 
cystidia (N. Menolli Jr. & M. Capelari, pers. comm.). 

The narrow septate elements in the pileipellis of P crenulatus are reminiscent 
of those typically exhibited by some members of sect. Hispidoderma (e.g., 
P. exiguus (Pat.) Sacc.). However the general organization of the pileipellis 
differs in those taxa and lacks the well-developed hymeniform layer made up 
of clavate or spheropedunculate elements that is typical of P stenotrichus and 
the other species with “Mixtini-type” pileipellis in sect. Celluloderma. 


Acknowledgments 

A. Justo acknowledges financial support from the NSF grant DEB0933081. The 
curators of MICH are gratefully acknowledged for sending the collection of Pluteus 
tucumanus on loan. Drew Minnis and Nelson Menolli Jr. provided very insightful 
comments in the presubmission reviews. 


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Justo A, Castro ML. 2007. Observations in Pluteus section Pluteus in Spain: Two new records for 
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Justo A, Vizzini A, Minnis AM, Menolli Jr N, Capelari M, Rodriguez O, Malysheva E, Contu M, 
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Justo A, Minnis AM, Ghignone S, Menolli Jr N, Capelari M, Rodriguez O, Malysheva E, Contu 
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Pluteus spp. nov. (Dominican Republic) ... 21 


morphology, geography and phylogeny. Mycol. Progress 10: 453-479. 
http://dx.doi.org/10.1007/s11557-010-0716-z 

Justo A, Caballero A, Mufioz G, Minnis AM, Malysheva E. 2011c. Taxonomy of Pluteus eugraptus 
and morphologically similar taxa. Mycologia 103: 646-655. http://dx.doi.org/10.3852/10-280 

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Soc. Mycol. France 72: 181-249. 

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Sinauer Associates, Sunderland, Massachusetts. 

Malysheva EF, Malysheva VF, Krasilnikova AA. 2009. Morphological and molecular approaches to 
study the genus Pluteus Fr. Mikol. Fitopatol. 43: 216-231. 

Matheny PB, Curtis JM, Hofstetter V, Aime MC, Moncalvo J-M, Ge ZW, Yang ZL, Sot JC, Ammirati 
J, Baroni TJ, Bougher NL, Hughes KW, Lodge DJ, Kerrigan RW, Seidl MT, Aanen DK, DeNitis 
M, Daniele GM, Desjardin DE, Kropp BR, Norvell LL, Parker A, Vellinga EC, Vilgalys R, 
Hibbett DS. 2006. Major clades of Agaricales: a multilocus phylogenetic overview. Mycologia 
98: 982-995. http://dx.doi.org/10.3852/mycologia.98.6.982 

Menolli Jr N, Asai T, Capelari M. 2010. Records and new species of Pluteus from Brazil based on 
morphological and molecular data. Mycology 1: 130-153. http://dx.doi.org/10.1080/21501203 
.2010.493531 

Midgley DJ, Saleeba JA, Stewart MI, Simpson AE, McGee PA. 2007. Molecular diversity of soil 
Basidiomycete communities in northern-central New South Wales, Australia. Mycol. Res. 
111: 370-378. http://dx.doi.org/10.1016/j.mycres.2007.01.011 

Minnis AM, Sundberg WJ. 2010. Pluteus section Celluloderma in the U.S.A. N. Amer. Fungi 
5: 1-107. http://dx.doi.org/10.2509/naf2009.005.001 

Munsell Color (ed.). 2009. Munsell Soil-Color Charts. Grand Rapids, Michigan. 

O’Brien HE, Parrent JL, Jackson JA, Moncalvo JM, Vilgalys R. 2005. Fungal community analysis 
by large-scale sequencing of environmental samples. Appl. Environ. Microbiol. 71: 5544-5550. 
http://dx.doi.org/10.1128/AEM.71.9.5544-5550.2005 

Orton PD. 1986. British fungus flora, agarics and boleti 4: Pluteaceae: Pluteus and Volvariella. Royal 
Botanic Garden, Edinburgh. 

Pegler DN. 1977. A preliminary Agaric flora of east Africa. Kew Bull., Additional Ser. 6: 1-615. 

Pegler DN. 1986. Agaric flora of Sri Lanka. Kew Bull., Additional Ser. 12: 1-519. 

Singer R. 1956. Contribution toward a monograph of the genus Pluteus. Transactions of the British 
Mycological Society 39: 145-232. http://dx.doi.org/10.1016/S0007-1536 (56)80001-6 

Singer R. 1958. Monograph of South American basidiomycetes, especially those of the east slope of 
the Andes and Brazil. Lloydia 21: 195-299. 

Stamatakis A, Hoover P, Rougemont J. 2008. A rapid bootstrap algorithm for the RAxML Web 
servers. Syst. Biol. 75: 758-771. http://dx.doi.org/10.1080/10635150802429642 

Vellinga EC. 1988. Glossary. Pp. 54-64, in: C Bas et al. (eds). Flora Agaricina Neerlandica 1. 
A.A. Balkema, Rotterdam. 

Vellinga EC. 1990. Pluteus Fr. Pp. 31-55, in: C Bas et al. (eds). Flora Agaricina Neerlandica 2. 
A.A. Balkema, Rotterdam. 

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http://dx.doi.org/10.3852/10-382 


ISSN (print) 0093-4666 © 2012. Mycotaxon, Ltd. ISSN (online) 2154-8889 


MY COTAXON 


http://dx.doi.org/10.5248/120.23 
Volume 120, pp. 23-33 April-June 2012 


The lichen genus Chapsa (Graphidaceae) in India 


SANTOSH JOSHI, DALIP K. UPRETI* & SANJEEVA NAYAKA 


Lichenology Laboratory, CSIR-National Botanical Research Institute 
Rana Pratap Marg, Lucknow - 226 001, India 
CORRESPONDENCE TO *: upretidk@rediffmail.com 


ABSTRACT — This paper provides a detailed account of the 16 species of Chapsa recorded in 
India. Chapsa patens and C. platycarpella are new records for the Indian lichen biota. 


Key worps — thelotremoid, distribution, identification key, Western Ghats 


Introduction 

Until recently the thelotremataceous lichen biota was limited in India to 
Chroodiscus, Diploschistes, Leptotrema, Myriotrema, Ocellularia, and Thelotrema 
(Awasthi 1991, 2000). Based on recent revisions (Frisch et al. 2006, Mangold et al. 
2009, Rivas Plata et al. 2010), Singh & Sinha (2010) cited 130 taxa representing 13 
genera in the Thelotremataceae Stizenb. for India. However, recent phylogenetic 
studies show that taxa previously classified in Thelotremataceae do not form 
a separate lineage but are nested within Graphidaceae. The Graphidaceae, 
containing the now-synonymous Thelotremataceae (Mangold et al. 2008), 
comprises the largest lichen family in India, where it is represented by 428 
species and 35 genera. 

Massalongo introduced Chapsa in 1860, a genus resurrected recently by 
Frisch et al. (2006) to incorporate species earlier classified in Thelotrema, 
Chroodiscus, Ocellularia, and Myriotrema. Chapsa is characterized by a thin, 
corticolous, endoperidermal to epiperidermal thallus, chroodiscoid ascomata 
with a fused to + free proper exciple, and periphysoids (Mangold et al. 2009). 
The closely related or similar Acanthotrema, Chroodiscus, and Reimnitzia differ 
mainly in spiny paraphysis tips, lack of periphysoids, and isidiate thallus with 
columnar crystalline layer, respectively. Moreover, Chroodiscus is typically 
foliicolous. Thelotrema and Topeliopsis differ mainly by lacking distinctly 
chroodiscoid ascomata. However, several species belonging to the last two 
genera require further investigation for proper delimitation and placement. 


24 ... Joshi, Upreti & Nayaka 


Due to their selective and preferred habitat, most members of thelotremoid 
Graphidaceae are restricted to lower altitudes in tropical to subtropical 
regions and thus inadequately understood (Hale 1974, 1978, 1981). As a 
group, thelotremoid lichens represent a significant component of corticolous 
rainforest microlichen biota, with some extending into temperate regimes 
(Santesson 1952; Sipman & Harris 1989; Rivas Plata et al. 2008; Purvis et al. 
1995). Chapsa is pantropical or subtropical but rarely temperate. Generally, 
it occurs below 1000 m and favors shaded microhabitats in rather disturbed 
old growth secondary forests (Rivas Plata et al. 2008). Chapsa flourishes in the 
southern and eastern regions of India, with some species found at altitudes of 
more than 2000 m. The dense moist rain forests of the eastern Himalayas and 
the evergreen forests of Western Ghats provide a suitable habitat for its growth. 
Most species of Chapsa grow on rough bark, thus indicating a particular type of 
forest community predominant in moist humid regions. The species generally 
acquire the same colour pattern as the substratum so that in the field many 
are difficult to distinguish from bark. The tree bark characteristics appear to 
influence the distribution patterns of the taxa in tree communities. Lichens 
growing on striate bark concentrate in deep furrows, with their ascomata 
emerging from cracks, while in many species the thallus surface mirrors inflated 
bark. Most taxa, however, grow on flaking bark with only ascomata protruding 
out of the thallus. 

The ascomatal features and chemical substances play a vital role in 
differentiating the different thelotremoid groups. Based on morphology, the 
ascomata may be of the chroodiscoid, fissurinoid, leprocarpoid, platycarpoid, 
lamelloid, scabioid, or topeliopsoid types. However, except for C. platycarpa 
and C. recurva, all Indian specimens lack secondary compounds. 

Sixteen species of Chapsa are currently known from India. The evergreen 
forests in the Western Ghats represented by nine species was the most diverse, 
with six species known from the tropical rain forests of the eastern Himalayas 
and four species known from the Andaman and Nicobar Islands. One report 
of Chapsa dilatata (Mull. Arg.) Kalb from Arunachal Pradesh (Dubey et al. 
2008, as Thelotrema dilatatum) was based on a misdetermined specimen that 
represents a Myriotrema species. All accurately identified Chapsa species 
recorded from India are presented here with brief descriptions. 


Materials & methods 

Material preserved in the herbarium of the National Botanical Research Institute 
(LWG) was examined morphologically, anatomically, and chemically. Thin hand-cut 
sections of apothecia and thallus mounted in tap water, cotton blue, 5% KOH, and iodine 
solution were observed under a compound microscope (LEICA DM 500). Chemical 
spot tests and TLC (using solvent system A) were conducted according to Orange et al. 
(2001). 


Chapsa in India... 25 


Taxonomic descriptions 


Chapsa alborosella (Nyl.) Frisch, Biblioth. Lichenol. 92: 90, 2006. 

The species is characterized by an ecorticate, pale olive green thallus lacking 
lichen compounds, round to angular or shortly elongate pale brown apothecia 
level with the thallus, 8-spored asci and hyaline, transversely septate, fusiform 
to clavate or oblong, I- ascospores. 

Chapsa platycarpella differs from the closely related C. alborosella in having a 
corticate thallus and a pale to blackish apothecial disc. Patwardhan & Kulkarni 
(1977a) and Nagarkar et al. (1988) reported this species from south India as 
Ocellularia alborosella. The species is common in tropical evergreen forest of 
Kerala in the Western Ghats. 


“Chapsa hiata” (Hale), ined. 

Known from evergreen forests in the southern part of India, this species is 
well characterized by an ecorticate thallus lacking lichen substances, wide and 
open apothecia with recurved margins, free proper exciple, 4—8-spored asci, 
and small hyaline muriform I- ascospores measuring 14-16 x 4-7um. 

Patwardhan et al. (1985) reported it from Karnataka as Thelotrema hiatum. 
Similar to Chapsa leprocarpoides, C. velata and C. pseudophlyctis in lacking 
thallus compounds, the species differs in having small ascospores. ‘The latter 
two species also differ in having a fused proper exciple. 


Chapsa indica A. Massal., Atti I. R. Ist. Veneto Sci. Lett. Arti, Ser. 3, 5: 257, 1860. 

This taxon is characterised by an ecorticate brownish-white to olive-grey 
thallus lacking lichen compounds, rounded to angular or shortly elongated and 
slightly branched apothecia that are level with the thallus, 6-8-spored asci, and 
hyaline transversely septate oblong-fusiform ascospores. 

Patwardhan & Nagarkar (1980) and Nagarkar et al. (1988) first reported the 
species from India (as Ocellularia pycnophragmia (Nyl.) Zahlbr.) from tropical 
rain forests of Andaman and Nicobar, Assam, and Meghalaya. Similar in having 
an ecorticate whitish (-brownish) to olive-grey thallus, C. leprocarpa and C. 
pseudophlyctis differ from C. indica in having muriform ascospores. 


Chapsa laceratula (Mill. Arg.) Rivas Plata & Licking, Lichenologist 42: 183, 2010. 

The species exhibits a pale to dark olive-green to olive brown, corticate 
surface lacking secondary metabolites, round to irregular, perithecioid to 
apothecioid apothecia with lacerate margins, 1-2(-4)-spored asci, and hyaline, 
muriform, fusiform ascospores. 

Patwardhan & Kulkarni (1977a) reported Chapsa laceratula (as Thelotrema 
laceratulum) from Karnataka, Kerala, Maharashtra, and Tamil Nadu in 
southern India. The scabioid apothecia with vertically layered excipulum and 


26 ... Joshi, Upreti & Nayaka 


<4-spored asci are the major distinguishing characters of C. laceratula, which 
morphologically may be confused with Topeliopsis species. It is generally 
distributed at lower elevation and growing luxuriantly in evergreen forests of 
Western Ghats. 
SPECIMENS EXAMINED: INDIA. KARNATAKA: Central Western Ghat, Shimoga district, 
3 km before Agumbe Ghat, from Koppa to Agumbe, alt. 649 m, on bark in evergreen 
forest, 14.01.2008, H.T. Lumbsch, D.K. Upreti, P.K. Divakar & J. Tandon 19730M/A 
(LWG); Uttarkannad, Sharavathi Ghat, near Gersoppa, alt. 718 m, on bark in evergreen 
forest, 16.01.2008, H.T. Lumbsch, D.K. Upreti, P.K. Divakar & J. Tandon 19744A-1, 
19744B, 1944C-2, 197441, 19745, 19745A-3, 19745C (LWG); Tamit Napvu: Madurai 
district, High ways Vattapara (Meghamaali), alt. 1580 m, on fallen twigs, 22.03.1999, S. 
Nayaka 92(LWG). 


Chapsa leprieurii (Mont.) Frisch, Biblioth. Lichenol. 92: 105, 2006. 

The species is characterized by a brown-olive, cartilaginous thallus lacking 
secondary compounds, rather small, rounded to slightly elongated or branched 
apothecia level with the thallus, 8-spored asci, and transversely septate brown 
oblong to ellipsoidal or clavate ascospores. 

Chapsa leprieurii has been reported from Karnataka, Kerala, and Tamil 
Nadu in southern India (Awasthi 1991, as Thelotrema leprieurii; Singh & Sinha 
2010, as T! leprieurii). The brown-spored similar species, C. platycarpa, differs 
in producing lichen compounds and a double margin. 


Chapsa leprocarpa (Nyl.) Frisch, Biblioth. Lichenol. 92: 108, 2006. 

The taxon exhibits a pale to dark olive-grey thallus lacking lichen compounds, 
large chroodiscoid apothecia with a lobed and recurved margin, pale brown 
to blackish but heavily white pruinose disc, 1-spored asci, and muriform 
ascospores. 

Awasthi (1991) first recorded C. leprocarpa (as Thelotrema colobicum) 
from Andaman and Nicobar Islands. Chapsa leprocarpa inhabits lowland and 
submontane regions in the Western Ghats. 

SPECIMENS EXAMINED: INDIA. KErAta: Idukki district, .C.R.I campus, Myladumpara, 
alt. ca. 1200 m, on bark of tree, 01.03.1984, D.D. Awasthi & G. Awasthi 84.88 (LWG- 
LWU); on way Myladumpara to Munnar, Santhampara area, alt. ca. 1200 m, on bark 


of tree, 02.03.1984, D.D. Awasthi & G. Awasthi 84.179 (LWG-LWU); Thikkady, Periyar 
Tiger Reserve, Sakunthalakadu, on bark, 23.03.2006, B. Haridas 06-009615/B (LWG). 


Chapsa leprocarpoides (Hale) Caceres & Liicking, Libri Bot. 22: 52, 2007. 

The species is ecorticate, pale olive to fawn coloured, lacking lichen 
substances, without compact surface, usually reflecting bark through the 
matted thallus surface; it has immersed to semi-immersed, rounded to angular 
apothecia with a flesh-coloured pruinose disc and low jagged to lobed margins, 
a pale to brownish or hyaline free proper exciple, 8-spored asci, and hyaline 
muriform ellipsoidal to oval ascospores. 


Chapsa in India ... 27 


Chapsa leprocarpoides was collected from evergreen tropical forests of 
Karnataka on rough tree barks at lower altitude. The ecorticate “Chapsa hiata” 
differs in having rather small ascospores while C. velata and C. pseudophlyctis 
have comparatively larger ascospores and a fused proper exciple. 


SPECIMENS EXAMINED: INDIA. KARNATAKA: Chikmagalure district, Chamudi Ghat, 
Kuvettu, alt. 104 m, on bark in evergreen forest, 15.01.2008, H.T. Lumbsch, D.K. Upreti, 
PK. Divakar & J. Tandon 19739R; Uttar Kannad, Sharavati Ghat, near Gersoppa, alt. 718 
m, on bark in evergreen forest, 16.01.2008, H.T. Lumbsch, D.K. Upreti, P.K. Divakar & 
J. Tandon 19744J (LWG); Shimoga district, Sagar to Talguppa, Ulanahalli, alt. 697 m, 
on bark in evergreen forest, 16.01.2008, H.T. Lumbsch, D.K. Upreti, P.K. Divakar & J. 
Tandon 19742E, 19742E-1(LWG); Central Western Ghat, Shimoga district, near Jog fall, 
Kargal, alt. 644 m, 16.01.2008, H.T. Lumbsch, D.K. Upreti, P.K. Divakar & J. Tandon 
19742B-2, 197430-1 (LWG). 


Chapsa meghalayensis (Patw. & Nagarkar) Lumbsch & Divakar, Lichenologist 42: 
183, 2010. 

This taxon is characterized by its ashy-white, ecorticate, granular thallus 
lacking lichen compounds, immersed and chroodiscoid apothecia with 
recurved margins, lacerate exciple, 2-spored asci, and small muriform brown 
ascospores measuring 27-46 x 11-16 um. 

First described from Meghalaya by Patwardhan & Nagarkar (1980) as 
Leptotrema meghalayense, the species was later transferred to Myriotrema by 
Awasthi (1991). Chapsa meghalayensis is endemic to India and collected from 
evergreen forests of northeast India. 


Chapsa patens (Nyl.) Frisch, Biblioth. Lichenol. 92: 111, 2006. PrP aA 

Thallus corticolous, epiperidermal, ecorticate, dark olivaceous grey to 
dark grey or blackish, continuous, + cracked due to bark texture, uneven, 
bark reflecting through thallus. Prothallus indistinct. Photobiont layer well 
developed, up to 50 um thick. Medulla endoperidermal. Apothecia scattered, 
sometimes grouped in two, immersed to level with thallus, mostly rounded to 
slightly angular or irregular in shape, up to 2.0 mm in diam. Margin raised, 
lobed to + continuous, mostly straight to recurved later, brownish to faintly 
crystalline pruinose inner side. Disc reddish-brown, exposed, covered by thick 
pruina. Proper exciple cupular, pale to hyaline, sometimes brownish, up to 40- 
80 um thick laterally. Periphysoides indistinct to 30 um long, inclined towards 
epihymenium. Epihymenium 10-15 um high, slightly brownish, granular. 
Hymenium, clear, 130-140 um high. Paraphyses entangled, simple, <1.5 
um thick, apically thickened, + dendroid branched, adspersed with grayish 
granules. Ascus, 1-spored, clavate, 90-130 x 25-40 um. Ascospores hyaline, 
densely muriform, with rounded ends, 80-125 x 20-35 um, I-. 

CHEMISTRY: K-, PD-, C-; no lichen compound detected in TLC. 

ECOLOGY & DISTRIBUTION: The species is a new record to India reported 
from tropical rain forests of the Eastern Himalayas where it is common in open, 


28 ... Joshi, Upreti & Nayaka 


canopied old forests with large tree trunks and generally found at 1700-2500 
m. Widely distributed and reported from Africa, Japan, and Sri Lanka. 

SPECIMEN EXAMINED: INDIA. WeEsT BENGAL: Eastern Himalaya, Darjeeling district, 

Tiger hill, north face of the hill, alt. ca. 2500 m, on bark of tree, 05.03.1967, D.D. Awasthi 

& M.R. Agarwal 67.21 (LWG-LWU). 
REMARKS: Chapsa leprocarpa, which also lacks lichen substances, differs from 
C. patens in having a slightly lower hymenium and smaller ascospores and 
grows at lower altitudes (1000-1200 m). The Indian specimen of C. patens 
exhibits a dirty grey to dark olivaceous thallus colour hardly distinguished 
from the bark, while C. leprocarpa has a pale-green to slightly greyish-green 
epiperidermal thallus. However, the hymenium and ascospores of the Indian 
sample are intermediate in size and closer to C. patens than C. leprocarpa and 
its distribution and thallus and apothecial morphology also agree well with 
the diagnostic features of C. patens. Hale (1981) and Matsumoto (2000) both 
described C. patens as a stictic acid-producing species that after re-investigation 
were shown to lack these substances. The records from Japan and Sri Lanka 
should be re-examined (Frisch et al. 2006). 


Chapsa platycarpa (Tuck.) Frisch, Biblioth. Lichenol. 92: 113, 2006. 

The species is recognized by the dark olive-green to olive-brown or pale 
yellowish-brown thallus containing the stictic acid chemosyndrome, a rounded 
to slightly irregular apothecioid to chroodiscoid shape in older material, 
erumpent apothecia with double margins, 8-spored asci, and transversely 
septate ellipsoidal to fusiform brown ascospores. 

Earlier placed in the neglected genus Phaeotrema due to its transversely 
septate brown ascospores, Chapsa platycarpa was reported from India (Kerala) 
as P. platycarpum (Patwardhan & Kulkarni 1977a). The similar C. leprieurii 
lacks stictic acid as a major thallus compound. 


Chapsa platycarpella (Vain.) Frisch, Biblioth. Lichenol. 92: 118, 2006. PL. 1B 

Thallus corticolous, endoperidermal, olive-green to pale-green, continuous, 
shiny, compact, cartilaginous, uneven to warty. Prothallus indistinct to 
brownish. Medulla white, indistinct, endoperidermal. Phenocortex well 
developed, dense, 20-30 um thick. Photobiont layer 20-35 um thick, with 
inclusion of calcium oxalate crystals, largely endoperidermal. Apothecia 
scattered to aggregate in groups, immersed to level with thallus, round to mostly 
angular or irregular in shape, <1.0 mm in diam. Margin thin, fissured to lobed, 
recurved, and yellowish to fawn with felty white pruinose to crystalline inner 
surface. Disc pale to blackish, distinctly white pruinose. Proper exciple cupular, 
pale to hyaline, 6-10 um wide. Periphysoides indistinct to 25 um long, inclined 
towards epihymenium. Epihymenium 5-8 um high, granular, unpigmented 


Chapsa in India ... 29 


to crystalline. Hymenium clear, 55-95 um high. Paraphyses simple, straight, 
<2.0 um thick, tips agglutinate with fine greyish to brownish granules. Ascus 
8-spored, clavate, 45-90 x 6-9 um. Ascospores, hyaline, transversely 3-5 
septate, fusiform with acute end cells, 12-16 x 3-5 um, I-. 
CHEMISTRY: K-, PD-, C-; no lichen compounds detected in TLC. 
ECOLOGY & DISTRIBUTION: Known to grow on smooth bark of young trees in 
lowland rainforests and coastal forests in shade as well as on exposed surfaces. 
In India, reported on trees in evergreen tropical forest in Kerala. 
SPECIMEN EXAMINED: INDIA. KERALA: Idukki, Adimali Forest Range, Thondi Kappu, 
on Myristica beddomei, 15.02.2006, B. Haridas 06-009598(LWG). 
REMARKS: Chapsa platycarpella, which is considered a separate species by Frisch 
et al. (2006), is otherwise treated as a synonym of C. alborosella. The species 
is well characterized and separated from the latter in having a cartilaginous 
thallus, thick phenocortex, and loosely aggregated apothecia with blackish 
discs covered by bluish-white pruina. 
Recently, Rivas Plata et al. (2010) placed the species in synonymy with 
C. astroidea (Berk. & Broome) Caceres & Liicking; however following Frisch 
(2006) with adequate distinguishing characteristics the species is accepted here 
as a new record for India. 


Chapsa pseudoexanthismocarpa (Patw. & C.R. Kulk.) Rivas Plata & Liicking, 
Lichenologist 42: 183, 2010. 

The taxon is characterized by a pale yellowish to greenish brown or pale olive 
thallus lacking secondary compounds, apothecia that are rounded to somewhat 
irregular and sometimes perithecioid, erumpent, immerged to distinctly 
emergent, 4-8-spored asci, and hyaline transversely septate bacilliform- 
fusiform ascospores. 

Patwardhan & Kulkarni (1977b) described the species from southern 
India (Kerala) in Ocellularia. Larger ascospores readily distinguish 
C. pseudoexanthismocarpa from C. indica. Other characters include the rough 
epiperidermal thallus, lepadinoid apothecia, and lacerate exciple. 


Chapsa pseudophlyctis (Ny1.) Frisch, Biblioth. Lichenol. 92: 120, 2006. 

The species is characterized by its white to whitish, whitish-grey to pale-grey 
thallus lacking secondary compounds, round to angular apothecia with jagged, 
often eroded margins having a white crystalline inner surface, 1-6-spored asci, 
and hyaline muriform ascospores. 

Chapsa pseudophlyctis may sometimes be confused with C. leprocarpa, which 
can be distinguished by asci consistently bearing a single larger ascospore. 
Jagadeesh Ram & Sinha (2009) first reported C. pseudophlyctis as new to India 
from Meghalaya and Sikkim. 


30 ... Joshi, Upreti & Nayaka 


PLATE. 1. New records of Chapsa from India. 
A. Chapsa patens. B. Chapsa platycarpella (note the apothecia). 
Scale bars: A = 2mm; B= 1 mm. 


Chapsa recurva (G. Salisb.) Frisch, Biblioth. Lichenol. 92: 95, 2006. 

Chapsa recurva and C. platycarpa are the only two Chapsa species that 
produce stictic acid as a major secondary compound (Rivas Plata et al. 2010). 
Nagarkar et al. (1986) reported the species (as Thelotrema recurvum) from 
Andaman & Nicobar Islands, but noted the absence of secondary compounds. 
Other major distinguishing characters include the corticate whitish grey smooth 
thallus, numerous solitary round to elongated semi-emergent chroodiscoid 
apothecia, and hyaline muriform ascospores. 


Chapsa in India... 31 


“Chapsa stellata” (Hale), ined. 

The taxon is recognized by the greenish, glaucous, continuous, mostly 
epiperidermal thallus lacking lichen substances, dispersed, slightly immersed 
chroodiscoid apothecia having a white pruinose disc and recurved margins, 
a + fused brown proper exciple, 1-spored asci, and ellipsoidal olivaceous to 
brownish muriform ascospores, 88-135 x 22-30 um. Patwardhan & Nagarkar 
(1980) reported the species (as Leptotrema stellatum) from Meghalaya in the 
eastern Himalayas. 


Chapsa velata (Nyl.) Caceres & Licking, Libri Bot. 22: 54, 2007. 

The species is characterized by the ashy-white smooth partly hypophloeodal 
thallus lacking lichen substances, semi-emergent chroodiscoid apothecia 
with erect to recurved margins, fused to free brown proper exciple, hyaline 
hymenium, 4-8-spored asci (1-2-spored according to Patwardhan & Nagarkar 
1980), and hyaline ellipsoidal muriform ascospores. The species was recorded 
from the Andaman Islands (Awasthi 1991, as Thelotrema velatum) and Nagaland 
(Patwardhan & Nagarkar 1980, as T. velatum). 


Key to Chapsa species recorded from India 


Ia. 2kSCOSPOrestirAalisVerselyesep latest. e8ih he Pst t tel Paik has chi doh |e Bloe ttl Le 2 
Ib AScosporest(Subs jot TITONIN «042. '3.q.4'¢ sane x poea 4 pean + po iat F paeaen sh sect eens iy 
2d HA SCOSPOKES NY AMIS cm, a Mwsere, ai eee AP ae ame Fld a Pt hae NP lo Oa ES LeeLee 3 
2D ASCOSPOLES’ DEO WAN: ET Si.e oped e potsate Zeon eh le eR lee eh cde Su eh SLOG CRs ty 6 
DT AASCOSPOLES 3s FOCI «ie creey Basecrary Basectery Banu hls Meank ly anohhy Tasneh ty Tatnat strat Casa haab Cisakate 4 
Sbt- ASCOSPOres <i AN MINIM. tela re x cosa .a ie 5 hls es wl laie adele 6 at wie Lae + ela oe Sule oa Sale eae S 
4a SAscospores (40=)770-120 uti lON SS. s.-..5%, cee oe a eaten atone abe wna C. indica 
4b. Ascospores 100-200 um long ...................000- C. pseudoexanthismocarpa 
5a. Ascospores 12-16 um long, 4—-6-septate, corticate................ C. platycarpella 
5b. Ascospores 17-22 um long, 5-9-septate, ecorticate................. C. alborosella 
6a. Stictic acid present, proper exciple free (double margin), 

apothecialteise-oreysbeowils $10.29 tt Soh id FLA ETA LAE Le C. platycarpa 
6b. Lichen substances absent, proper exciple fused, 

dise. pale: brown 10 CrevISh nthe ni dai sebding at feed bata hah did C. leprieurii 
PAPASCOSPOFESINV ANI Sot inches Bevcttes Besmartes and, ao Als Mie Al Ware Als Taree idee een a eee 8 
7D ASCOSPOFES, DEO WM yc lela + satel ss coe wx + kelses 5 ablahe boat de blots oy Blas om als adele oie 5 
BAVA SCOSPOKES: OU MIT LOUS ets irate I cate I pelt bedded on etn te ata heat nc er ne 9 
Shi Ascosporesi= GO (Monge: chin peahies pesca ok wha eh ieee ie he de’ ye § 13 
9a. Ascospores 12-15 um long, lichen substances absent .................. “C. hiata” 


Ob: -Ascosporess-l oyun lores chemastryavariabl ess. tant gi, fact ice died Pct fds Hie ded tga dee ood 10 


32 ... Joshi, Upreti & Nayaka 


10a, Sticheacid ‘presents cu 25 G0.2. 2400. Vain Gite aae eine yale C. recurva 
ONT Le CTE, SUIS EAIICR SAI SOITE cas Ped ais esd Sank Ue ad iG dO cece A cat IE deh 11 
lla. Proper exciple free, apothecia often aggregate, 

ascospores GOES RALO= 212 WTTe: +. eats! thera ven More woh Save aby baragee hand C. leprocarpoides 
11b. Proper exciple fused apothecia solitary, ascospores 30-60 x 12-20 um......... 12 
12a. Thallus ashy-white, apothecial disc dark-grey, thinly pruinose 

PvE ONES SEITE Cea eau en des che te ha ka Md cA Wak Le C. velata 
12b. Thallus pale-olive, apothecial disc pale brown, 

distinctly white prUinoSe am a: garg wh he gs gs ed aig Seal de C. pseudophlyctis 
13a. Thallus with prosoplectenchymatous cortex, proper exciple layered C. laceratula 
13 b wlnalhisecortiGaie. ng OP us. t-ratcas, ke te ee Re we 2S A gE EO eRe ce 14 
14a. Ascospores. 2125, pny lone: oe eee es eet mee BM ene BS ane OF matey hoe enone C. patens 
146 ASCOSPORES FO PMIONG 1.55 f.t.08 fel EF eee wel! we wee as C. leprocarpa 
15a. Ascospores 2746-56 1 U6 filth acts, sctee aces ots eg Gis st oe C. meghalayensis 
15b: AScospores:$8= 135562 2—S Oui x sree, stirs gills ies tect tlie ey tetliredey 9 alii “C. stellata” 


Acknowledgements 

We are grateful to the Director of the CSIR-National Botanical Research Institute, 
Lucknow, for providing necessary facilities, and the Ministry of Environment and 
Forests, New Delhi, for financial support. The authors are also grateful to Dr. R. Liicking 
and Dr. Pradeep. K. Divakar for their valuable comments on manuscript. 


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ISSN (print) 0093-4666 © 2012. Mycotaxon, Ltd. ISSN (online) 2154-8889 


MYCOTAXON 


http://dx.doi.org/10.5248/120.35 
Volume 120, pp. 35-41 April-June 2012 


A new species of Inonotus (Hymenochaetaceae) and 
Trametes cingulata (Polyporaceae) newly recorded from Brazil 


MaAtrA CORTELLINI ABRAHAO & ADRIANA DE MELLO GUGLIOTTA 


Instituto de Botanica, Nucleo de Pesquisa em Micologia 
Caixa Postal 68041, CEP 04045-972, Sao Paulo, SP. Brazil 
* CORRESPONDENCE TO : mairaabrahao@hotmail.com 


ABSTRACT — Two interesting species found during a survey of polypore fungi in northwestern 
Sao Paulo State, Southeast Brazil, are described and illustrated. Inonotus multisetifer is 
proposed as a new species characterized by resupinate basidiomata with round to angular 
pores, 6—9 per mm, acute setal hyphae embedded in trama, subulate hymenial setae, and 
globose to subglobose basidiospores. Trametes cingulata constitutes a first record from 
Brazil. 


KEY worDs — mycodiversity, neotropics, Polyporales, Hymenochaetales 


Introduction 

The Atlantic Rain Forest, which has 20,000 species of plants of which 6000 
are endemic, holds today less than 8% of its original extent in Brazil and has 
been rated as one the world’s top five biological hotspots (Mittermeier et al. 
1999, SOS Mata Atlantica/INPE 2009). 

The state of Sao Paulo is located in southeastern Brazil and has an area of 
248,808.8 km’. Mostly inserted in the Atlantic Forest domain (68%), the state 
contains remnants of rain forest, Araucaria forest, seasonal semideciduous 
forest, transitions between them, and remnants of vegetation of restinga and 
mangrove (Kronka 2005, SOS Mata Atlantica/INPE 2009). 

Currently, the natural vegetation cover of the state amounts to only 13.94% 
of its surface (Kronka 2005, Nalon et al. 2008), concentrated mainly along the 
coastal zone, where there is a concentration of natural areas of Integral Protection 
(Xavier et al. 2008). In the northwest region, only minor conservation units are 
found (Kronka 2005, Xavier et al. 2008), with the majority of the remaining 
forest located in private areas where their conservation is key to biodiversity 
conservation in the State. 


36 ... Abrahao & Gugliotta 


The mycodiversity in northwest Sao Paulo State is almost unknown; 
currently, only 36 species of basidiomycetes have been cited from this region 
(Xavier-Santos 2003, Abrahao et al. 2008, 2009). 

As part of a wider project dealing with biodiversity of remnant forest 
fragments of the northwest region of Sao Paulo State, this study reports a 
new species of Inonotus and a new record of Trametes cingulata from Brazil, 
including descriptions, illustrations, and discussions on the species. 


Materials & methods 

Collections were performed between March 2007 and December 2008 in 
Semidecidual Stacional Forest fragments in northwest Sao Paulo State, Brazil. 

The material was studied following the classical methods for polypores (Gilbertson 
& Ryvarden 1986, Ryvarden 1991, 2004). Micromorphological observations were made 
from material mounted in 5% KOH and Melzer’s reagent; measures were made in 5% 
KOH in a Leica DM 1000 microscope. 

Vouchers are deposited in SP, SJRP, and O (acronyms follow Holmgren & Holmgren 
1998). 

Nomenclature and classification systems follow those of Kirk et al. (2008), 
Centraalbureau voor Schimmelcultures (www.cbs.knaw.nl), and Index Fungorum 
(www.indexfungorum.org). 


Taxonomy 


Inonotus multisetifer Abrahao & Gugliotta, sp. nov. Figs 1-4 
MycoBank MB 563746 
Differs from Inonotus adnatus in larger basidiomata, slightly narrower setal hyphae, 


smaller scant hymenial setae, and larger (8.8-11.2 x 8.8-10 um) globose to subglobose 
basidiospores. 


Type: BRAZIL. Sado Paulo State: Sdo Jodo de Iracema, SAo Francisco Farm, G5 area 
(20°28'25"S 50°17'36"W), 17.IV.2007, M.C. Abrahao 270 (holotype, SP393791; isotype, 
SJRP). 


ErymMo_ocy: Referring to the abundant setal hyphae. 


BASIDIOMA annual, resupinate, up to 8 cm in diameter, ca. 4 mm thick, strongly 
adnate, hard and dense when dry. MarGaIn thin to absent. PORE SURFACE 
ochraceous to brown, pores round to angular, 6—9 per mm, invisible to the 
naked eye; tubes dark brown, up to 4 mm deep. CONTEXT homogeneous, very 
thin, virtually absent in parts, cinnamon. HYPHAL SYSTEM monomitic, IKI-; 
generative hyphae simple-septate, thin to thick-walled, golden to rusty brown, 
(2.5—)3.75 um wide. SETAL HYPHAE abundantly present, dark brown, acute, 
thick-walled, 100-200 x 8.75—16.25 um, embedded in trama and not projecting. 
HYMENIAL SETAE scant and in some sections totally absent, apparently found 
only in the young pores and in the pores mouths, dark brown, subulate, thick- 
walled, 33.75—45 x 8.75-10 um. Basrpt1a clavate, hyaline, with four sterigmata, 


Inonotus multisetifer sp. nov. (Brazil) ... 37 


FiGuRES 1-5 (scale bars = 10 um). Fics. 1-4: Inonotus multisetifer (SP393791, holotype) 
— 1, basidiospores; 2, basidia and immature basidiospore; 3, hymenial setae; 4, setal 
hyphae. Fic. 5. Trametes cingulata (SP416154) — basidiospores. 


22.5—23.75 x 5—6.25 um. Basip1osPorREs globose to subglobose, IKI-, slightly 
thick-walled, hyaline to pale yellow, 8.75-11.25 x 8.75-10 um. 

SUBSTRATE & DISTRIBUTION —dead hardwood; known only from the type 
locality in Brazil. 


38 ... Abrahaéo & Gugliotta 


ADDITIONAL SPECIMENS EXAMINED —Inonotus adnatus: COSTA RICA. PUNTARENAS: 

La Amistad Pacifico, Estacion Progreso, near the station, alt 1180 m, 27.1X.2000, 

L. Ryvarden 42795 (0316222-079, isotype). Inonotus micantissimus: BRAZIL. R1o 

GRANDE DO SUL STATE: Sao Salvador, III.1944, S.J. Rick 20691 (PACA8878, holotype). 

Inonotus pegleri: TANZANIA. EASTERN PROVINCE: KiLosa District, Mikumi 

National Park, on dead fallen tree, alt 487 m, 30.IV.1968, D.N. Pegler T725 (K167869, 

holotype; 0296, isotype). 
ComMENTSs — Inonotus multisetifer is characterized by resupinate basidiomata, 
presence of acute setal hyphae in the trama, scant subulate hymenial setae, and 
globose to subglobose slightly thick-walled hyaline to pale yellow basidiospores. 
This combination of characters relate this species to other Inonotus resupinate 
species that also have setal hyphae, hymenial setae, and basidiospores varying 
from globose, subglobose to ellipsoid: I. adnatus Ryvarden, I. micantissimus 
(Rick) Rajchenb., and I. pegleri Ryvarden. ‘The main diagnostic characteristics 
are shown in TABLE 1. 


TABLE 1. Key features of Inonotus multisetifer and morphologically related species. 


I. multisetifer I. adnatus I. micantissimus I. pegleri 
Basidioma <8cm diam. <4cm diam. 10x4cm 12cm 
(thickness) (~4 mm) (~3 mm) (<14 mm) (< 15 mm) 
Pores/mm 6-9 6-9(-10) 5-6 4-6 
Generative hyphae: Thin to thick; Thin to thick; — Slightly thick; Thin to thick; 
walls; golden to golden to yellowish to chestnut hyaline to 
color rusty brown rusty brown light-brown 
Hymenial setae 33.8—-45 x 26.2—-56.2 x 22.5-27.5 X 20-21.2 x 
(um) 8.8—10 10-12.5 6.2-11.2 6.2-8.8 
Setal hyphae 100-200 x < 150 x 160-300(-400) x 125-520 x 
(um) 8.8—16.2 8.8-17.5 8.8-20* 7.5-21.2 
Basidiospore shape; (Sub) globose; Globose; (Sub)globose, (apiculate); | Globose; 
color hyaline to yellowish hyaline to pale melleous hyaline to 
yellowish yellowish 
Basidiospore size 8.8-11.2 x 7-8.8 diam. 11.25 x 8.8-10 6-7 um wide 
(um) 8.8-10 


*(width up to 25 um in KOH) 


Inonotus multisetifer is morphologically most closely related to I. adnatus, 
a species known only from the type locality in Costa Rica (Ryvarden 2002, 
2004, 2005). Inonotus adnatus presents a smaller basidioma, slightly wider 
setal hyphae, larger and ventricose hymenial setae, and smaller globose 
basidiospores. Hymenial setae are abundant in the type specimen of I. adnatus 
but scant to mostly absent in I. multisetifer. 


Inonotus multisetifer sp. nov. (Brazil) ... 39 


Inonotus micantissimus, which also occurs in Brazil (Rajchenberg 1987, 
Baltazar et al. 2010), has larger pores, longer setal hyphae, smaller hymenial 
setae, and basidiospores with abundant oily contents. 

Inonotus pegleri is easily separated by the combination of larger pores, 
smaller hymenial setae, longer setal hyphae, smaller basidiospores, and its 
limited distribution in Africa (Ryvarden 2005). 


Trametes cingulata Berk., Hooker’s J. Bot. Kew Gard. Misc. 6: 164, 1854 FIG. 5 

BASIDIOMA annual, lignicolous, sessile pileate to pseudostipitate, dimidiate, 
many basidiomata from one stipe or solitary; pileus semicircular, applanate 
and thinner near the margin, with contracted base, soft when fresh and flexible 
when dry, 0.7—4.6 x 0.7—3.9 x 0.1—0.8 cm. PILEAR SURFACE in brown and cream 
tints, concentrically zonate, glabrous. MarGIN round, concolorous with the 
pilear surface, entire, glabrous and sterile. Stipe when present circular, cream 
to brown, glabrous, dense, 0.2-1.1 x 0.2-0.9 x 0.5-1.3 cm. PORE SURFACE 
poroid, lighter than pilear surface, pores angular to elongated, 7-9 per mm; 
tubes stratified, forming up to 4 distinct layers, light-brown, up to 2 mm deep; 
dissepiments thin to slightly thick, lacerate. CONTEXT duplex, the upper layer 
fibrous, greyish to blackish, thin, at least at the base separated by a thin black 
line from the lower part, the latter light-brown, corky, up to 6 mm thick at 
the base, negative reaction in KOH. HypHaAt sysTeEM trimitic, IKI-; generative 
hyphae hyaline, with clamps, thick-walled, 2.5—-3.75 um wide; skeletal hyphae 
yellowish, thick-walled, abundant in the basidioma, golden and solid in the 
context, 3.75—6.25 um wide; binding hyphae hyaline to yellow, thick-walled to 
semisolid in the context, slightly irregular and moderately branched, frequent, 
1.25-3.75 um wide. Cystrip1a absent. Basrp1a clavate, hyaline, thin-walled, 
11.25-12.5 x 3.75—5 um. Basip10sPorEs obovoid, hyaline, IKI-, smooth, thin- 
walled, 5—6.25 x (2.5—)3.75 um. 

SUBSTRATE & DISTRIBUTION — causing white-rot in twigs of dead angiosperm. 
Previously cited from Asia, Africa, and Australia (Ryvarden & Johansen 1980, 
Young et al. 2004, Gilbert et al. 2008) and Costa Rica and Venezuela in America 
(Ryvarden & Iturriaga 2001, Mata et al. 2007). 


MATERIAL EXAMINED — BRAZIL. SAO PAULO STATE: Palestina, Boa Vista Farm, P6 
area (20°19'16"S 49°30'17”W), 10.XII.2008, A.M. Gugliotta 1410 (SP416154, SJRP); Sao 
Joao de Iracema, Sao Francisco Farm, G5 area (20°28'25"S 50°17'36"W), 27.XI.2007, 
M.C. Abrahao 319 (SP393683). INDIA. BEHAR: Soane River [BIHAR: Son River], on 
dead wood, J. Hooker s.n. (K167868, isotype). 


COMMENTS — Macroscopically the species may be recognized by its glabrous 
basidioma in brown and cream tints (Ryvarden & Johansen 1980). The angular 
to elongated pores, duplex context with a greyish to blackish fibrous upper 


AO ... Abrahdo & Gugliotta 


layer, separated by a thin black line from the lower light-brown corky part, 
and obovoid, hyaline, thin-walled basidiospores support the identification. It 
constitutes a new record from Brazil. 


Acknowledgments 

The authors acknowledge Dr. Leif Ryvarden for identification of Trametes 
cingulata (SP393683). We are grateful to FAPESP (first author's fellowship process n° 
2006/58786-6 and BIOTA thematic project process n° 2004/04820-3) for financial 
support. We also extend our thanks to Gerardo Lucio Robledo and Rosa Mara Borges 
da Silveira, who kindly reviewed the manuscript. 


Literature cited 

Abrahao MC, Gugliotta AM, Silva R, Fujieda RJY, Boscolo M, Gomes E. 2008. Ligninolytic activity 
from newly isolated basidiomycete strains and effect of these enzymes on the azo dye orange II 
decolourisation. Annals of Microbiology 58: 427-432. 

Abrahao MC, Gugliotta AM, Gomes E. 2009. Polipordides (Basidiomycota) em fragmentos de mata 
no perimetro urbano de Sao José do Rio Preto, Sao Paulo, Brasil. Revista Brasileira de Botanica 
32: 427-440. http://dx.doi.org/10.1590/S0100-84042009000300004 

Baltazar JM, Trierveiler-Pereira L, Ryvarden L, Loguercio-Leite C. 2010. Inonotus s.l. 
(Hymenochaetales) in the Brazilian herbaria FLOR and SP. Sydowia 62: 1-9. 

Gilbert GS, Gorospe J, Ryvarden L. 2008. Host and habitat preferences of polypore fungi in 
Micronesian tropical flooded forests. Mycological Research 112: 674-680. http://dx.doi. 
org/10.1016/j.mycres.2007.11.009 

Gilbertson RL, Ryvarden L. 1986. North American polypores. Synopsis Fungorum 1: 1-433. 

Holmgren PK, Holmgren NH. 1998. Index Herbariorum: New York Botanical Garden's Virtual 
Herbarium. Available at: http://sweetgum.nybg.org/ih/Holmegren. 

Kirk PM, Cannon PF, Minter DW, Stalpers J. 2008. Ainsworth & Bisby’s dictionary of the fungi. 
10th ed. Wallingford, CAB International. 771 p. 

Kronka FJN. 2005. Inventario florestal da vegetacao natural do Estado de Sao Paulo. Sao Paulo, 
Secretaria de Meio Ambiente/Instituto Florestal. 200 p. 

Mata M, Ruiz-Boyer A, Carranza J, Ryvarden L. 2007. Nuevos registros de hongos poliporoides 
(Basidiomycetes) para Costa Rica. Boletin de la Sociedad Micoldgica de Madrid 31: 123-129. 

Mittermeier RA, Myers N, Gil PR, Mittermeier CG. 1999. Hotspots: earth’s biologically richest and 
endangered terrestrial ecoregions. Ciudad de Mexico, CEMEX/Agrupacion Sierra Madre. 430 
p. 

Nalon MA, Mattos IFA, Franco GADC. 2008. Meio fisico e aspectos da fragmentacao vegetal. 
15-21, in: Rodrigues RR, Bononi VLR (Orgs.). Diretrizes para a Conservacao e Restauracao da 
Biodiversidade no Estado de Sao Paulo. Sao Paulo, Instituto de Botanica. 

Rajchenberg M. 1987. Type studies of Polyporaceae (Aphyllophorales) described by J. Rick. Nordic 
Journal of Botany 7: 553-568. http://dx.doi.org/10.1111/j.1756-1051.1987.tb02023.x 

Ryvarden L. 1991. Genera of polypores. Nomenclature and taxonomy. Synopsis Fungorum 5: 
1-363. 

Ryvarden L. 2002. Studies in neotropical polypores 17. New neotropical Inonotus species. Synopsis 
Fungorum 15: 70-80. 

Ryvarden L. 2004. Neotropical polypores. Part 1. Introduction, Ganodermataceae & 
Hymenochaetaceae. Synopsis Fungorum 19: 1-227. 


Inonotus multisetifer sp. nov. (Brazil) ... 41 


Ryvarden L. 2005. The genus Inonotus - a synopsis. Synopsis Fungorum 21: 1-149. 

Ryvarden L, Iturriaga T. 2001. Studies in neotropical polypores 9. A critical checklist of poroid 
fungi from Venezuela. Mycotaxon78: 393-405. 

Ryvarden L, Johansen I. 1980. A preliminary flora of East Africa. Fungiflora: Oslo, 636p. 

SOS Mata Atlantica, Instituto Nacional de Pesquisas Espaciais. 2009. Atlas dos remanescentes 
florestais da Mata Atlantica, Periodo 2000 a 2005. Available at: http://www.sosma.org.br. 

Xavier AF, Bolzani BM, Jordao S. 2008. Unidades de conservacao da natureza no Estado de 
Sao Paulo. 23-42, in: Rodrigues RR, Bononi VLR (Orgs.). Diretrizes para a Conservacao e 
Restauracao da Biodiversidade no Estado de Sao Paulo. Sao Paulo, Instituto de Botanica. 

Xavier-Santos S. 2003. Isolamento, identificagao e perfil enzimatico de fungos decompositores de 
madeira da Estagao Ecoldgica do Noroeste Paulista —- Sao José do Rio Preto/Mirassol, SP. 222f. 
PhD Thesis (Ciéncias Biolégicas, Microbiologia Aplicada), Universidade Estadual Paulista, Rio 
Claro. 

Young AM, Fechner NA, Ryvarden L. 2004. A preliminary checklist and introductory notes on the 
macrofungi of Lamington National Park. Australasian Mycologist 23: 45-52. 


ISSN (print) 0093-4666 © 2012. Mycotaxon, Ltd. ISSN (online) 2154-8889 


MY COTAXON 


http://dx.doi.org/10.5248/120.43 
Volume 120, pp. 43-48 April-June 2012 


Manoharachariella indica sp. nov. from the Western Ghats, India 


KUNHIRAMAN C, RAJESHKUMAR' & SANJAY K. SINGH? 


National Facility for Culture Collection of Fungi, 
MACS’ Agharkar Research Institute, G.G. Agarkar Road, Pune, India 
CORRESPONDENCE TO: ‘rajeshfungi@gmail.com & *singhsksingh@gmail.com 


ABsTRACT — Manoharachariella indica is described as a new species from India, differing 
from M. lignicola, the only other described species of the genus, by larger conidia, longer 
conidiophores, and additional conidiophore septa. 


KEY worpDs — anamorphic fungi, dematiaceous hyphomycete, Tamhini Ghats, Maharashtra 


Introduction 

The Western Ghats, one of the world’s most diverse ‘biodiversity hot 
spots, is unique and diverse for its fungal wealth. Studies on microfungi and 
exploration of fungal diversity from different microhabitats in the Western 
Ghats are routinely conducted by the National Fungal Culture Collection of 
India (NFCCI) (Rajeshkumar et al. 2010, 2011a, b; Singh et al. 2009, 2010). 
During a September 2011 expedition to explore microfungal diversity in the 
Mulshi and adjoining areas we discovered a dematiaceous hyphomycete on a 
dead twig from the bamboo dominating the evergreen patch in the valley of the 
Tamhini Ghats. 

Bagyanarayana et al. (2009) recently established Manoharachariella for 
M. lignicola Bagyan. et al. from litter in Andhra Pradesh, India. Monoblastic 
integrated conidiogenous cells that produce solitary doliiform obpyriform 
dictyoseptate apiculate conidia characterize this dematiaceous hyphomycete 
genus. We describe a second species in this genus. 


Materials and methods 

ISOLATES AND MORPHOLOGY — Conidia were isolated directly from the surface of 
a dead twig and observed under a Nikon binocular stereomicroscope (Model SMZ- 
1500 with Digi-CAM, Japan). Single conidial cultures were established on 2% potato 
dextrose agar plates (PDA; Crous et al. 2009). For morphotaxonomic studies and 
photomicrographs, Zeiss (AXIO Imager 2, Germany) and Olympus (Model CX-41, 


44 ... Rajeshkumar & Singh 


Japan) microscopes were used. Conidia and conidiophores were mounted in lactic acid 
cotton blue and measured using an ocular micrometer (and confirmed with software 
available with the Zeiss microscope), with 30 observations per structure. Culture colony 
characteristics were studied on two different media: 2% malt extract agar (MEA) and 
PDA (Crous et al. 2009). Herbarium specimens are deposited in the Ajrekar Mycological 
Herbarium (AMH), and the culture NFCCI 2630 was accessioned and preserved at 
NFCCI; WDCM-932, Agharkar Research Institute, Pune, India. 


Taxonomy 


Manoharachariella indica Rajeshkumar & S.K. Singh, sp. nov. PLaTEs 1-3 
MycoBank MB 563453 
Differs from Manoharachariella lignicola by larger conidia, longer conidiophores, and 


additional conidiophore septa. 


Type: India, Maharashtra, Tamhini Ghats, on dead twigs, September 2011, K.C. 
Rajeshkumar (Holotype, AMH 9438; ex-type culture NFCCI 2630). 


EryMoOLoey: indica, referring to the country where this fungus is native. 


Colonies effuse, thin, mid to dark brown or blackish brown, mycelium superficial 
or semi immersed or immersed, hyphae pale to dark brown, smooth, 4-6 um. 
Stroma none. Setae and hypopodia absent. Conidiophores macronematous, 
mononematous, straight or flexuous, sparingly branched, arising laterally and 
apically from the immersed mycelium, 5—15-septate, smooth, pale to dark brown 
76-276 x 6-10.8 um. Conidiogenous cells monoblastic, integrated, terminal or 
yw a con Haare — 


Fic. 1. Manoharachariella indica (holotype): a. Habit. b. Sticky conidia in false chain. c. Terminal 
and lateral conidiogenesis. d—f. Conidiophores and conidia in nature. 


Manoharachariella indica sp. nov. (India) ... 45 


SO ‘woz 


Fig. 2. Manoharachariella indica (holotype): a. Conidial development. b-c. Apiculate conidia. 
d,f-g,j-k. Variation in conidial size and shape. e. Conidia with conidiophores. h. Conidia under 
phase contrast view. i. Sticky conidia in false chain. 


46 ... Rajeshkumar & Singh 


Fic. 3. Manoharachariella indica (holotype): a-b. Mature conidiophores. c. Young conidiophores. 
d. Hyphal branching and initiation of conidiophores in nature. e. Conidiophores and attached 
conidia. f. Conidiophores with young conidia. g. Foot cells of conidiophores. h. Mature conidia 
attached with conidiophores, showing multi-polar germination. i. Branched conidiophore. j. 


Simple conidiophore. 


Manoharachariella indica sp. nov. (India) ... 47 


intercalary, pale to mid brown, 4.4-8.6 x 4-7.6 um. Conidia solitary, dry or 
sticky, acropleurogenous, simple, doliiform, obpyriform, ellipsoidal or irregular, 
apiculate, apiculus rounded or obtuse, smooth, dictyoseptate, longitudinally 
10-13-septate, tiered, pale to dark brown or blackish brown, apical and basal 
tiers hyaline or subhyaline. 84-110(-120) x 31-44 um; young conidia hyaline 
or subhyaline, 56 x 17 um. 

Conidia in nature are dry and/or sticky and also form false chains of 2-3 
conidia. Conidial germination was observed as being multi-polar in moist 
chambers, but also after 12 hours of inoculation on PDA plates. 

Colonies on PDA and MEA very slow growing, 1-2 mm diam after 14 days, 
pale brown, velutinous; reverse pale brown. 

TELEOMORPH: not observed. 


Discussion 

The monotypic Manoharachariella with its type species M. lignicola 
somewhat resembles Septosporium Corda (especially S. rostratum M.B. Ellis, 
1961) in having dictyoseptate beaked conidia but differs in the absence of 
setae, unbranched conidiophores, and percurrent conidiogenous cells. Two 
species of Xenosporium Penz. & Sacc. — X. africanum Piroz. (Deighton 
& Pirozynski 1966) and X. boivinii S. Hughes (Hughes 1978)— resemble 
Manoharachariella in conidial shape but differ in their monoblastic, acrogenous 
or acropleurogenous, obpyriform, apiculate, smooth and dictyoseptate conidia 
that are transversely longitudinally septate. In addition, secondary conidia are 
absent in Manoharachariella but present in Xenosporium. Manoharachariella, 
like Bioconiosporium Bat. & J.L. Bezerra (Ellis 1976), produces solitary 
dictyoseptate conidia but the conidia are monoblastic with single apiculus 
and lack the two large protuberances that are prominent in Bioconiosporium. 
Conidia in both Monodictys S. Hughes (Ellis 1971) and Manoharachariella are 
monoblastic, dictyoseptate, and acrogenous but those in Manoharachariella 
are apiculate and tiered. Bagyanarayana et al. (2009) noted all these differences 
when they established Manoharachariella. 

Manoharachariella lignicola is differentiated from M. indica by its smaller 
(42.5-50.5 x 25-32 um) 7-9 transversely septate conidia and smaller 
conidiophores (< 35 um x 3-4.5 um) with fewer (1-3) septa. 


Acknowledgements 

We are indebted to Pedro W. Crous (Centraalbureau voor Schimmelcultures, Utrecht, 
The Netherlands) and Uwe Braun (Martin-Luther-University, Halle, Germany) for 
commenting on this manuscript. Thanks are also due to the Department of Science and 
Technology (DST), Government of India, New Delhi for providing financial support for 
setting up the National Facility for Culture Collection of Fungi (No. SP/SO/PS-55/2005) 
at MACS’ Agharkar Research Institute, Pune, India, and the Director, MACS’ ARI for 
providing facility. 


48 ... Rajeshkumar & Singh 


Literature cited 

BagyanarayanaG, Rao NK, KunwarIK.2009. Manoharachariella,anew dematiaceoushyphomycetous 
genus from India. Mycotaxon 109: 301-305. http://dx.doi.org/10.5248/109.301 

Crous PW, Verkley GJM, Groenewald JZ, Samson RA (eds). 2009. Fungal Biodiversity. CBS 
Laboratory Manual Series. Centraalbureau voor Schimmelcultures, Utrecht, Netherlands. 

Deighton FC, Pirozynski KA. 1966. Microfungi II. Brooksia and Grallomyces; Acrogenotheca ornata 
sp. nov.; the genus Xenosporium. Mycol. Pap. 105. 35 p. 

Ellis MB. 1961. Dematiaceous hyphomycetes. III. Mycol. Pap. 79. 23 p. 

Ellis MB. 1971. Dematiaceous hyphomycetes. CMI, Kew, UK. 608 p. 

Ellis MB. 1976. More dematiaceous hyphomycetes. CMI, Kew, UK. 507 p. 

Hughes SJ. 1978. New Zealand fungi. 25. Miscellaneous species. N. Z. J. Bot. 16(3): 311-370. 

Rajeshkumar KC, Singh PN, Yadav LS, Swami SV, Singh SK. 2010. Chaetospermum setosum sp. nov. 
from the Western Ghats, India. Mycotaxon 113: 397—404. http://dx.doi.org/10.5248/113.397 

Rajeshkumar KC, Hepat RP, Gaikwad SB, Singh SK. 201 1a. Pilidiella crousii sp. nov. from northern 
Western Ghats, India. Mycotaxon 115: 155-162. http://dx.doi.org/10.5248/115.155 

Rajeshkumar KC, Sharma R, Hepat RP, Swami SV, Singh PN, Singh SK. 2011b. Morphology and 
molecular studies on Pseudocercospora kamalii sp. nov. a foliar pathogen on Terminalia from 
India. Mycotaxon 117: 227-237. http://dx.doi.org/10.5248/117.227 

Singh SK, Singh PN, Yadav LS, Hepat RP. 2009. A new species of Gonatophragmium from Western 
Ghats, India. Mycotaxon 110: 183-187. http://dx.doi.org/10.5248/110.183 

Singh SK, Yadav LS, Singh PN, Sharma R, Rajeshkumar KC. 2010. Anewrecord of Gliocephalotrichum 
(Hypocreales) from India. Mycotaxon 114: 163-169. http://dx.doi.org/10.5248/114.161 


ISSN (print) 0093-4666 © 2012. Mycotaxon, Ltd. ISSN (online) 2154-8889 


MY COTAXON 


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Volume 120, pp. 49-58 April-June 2012 


Russula jilinensis sp. nov. (Russulaceae) from northeast China 


Guo-ji£ Li»?, SAt-Fer Li ?, XING-ZHONG Liu! & HUA-AN WEN ™ 


'State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, 
No3 'st West Beichen Road, Chaoyang District, Beijing 100101, China 

*Graduate University of Chinese Academy of Sciences, Beijing 100049, China 

* CORRESPONDENCE TO: wenha@im.ac.cn 


ABSTRACT — Russula jilinensis (subg. Coccinula sect. Laetinae), is described from Changbai 
Mountains, northeast China. The new species is distinguished by its bright red glabrous pileus 
with a cinnamon tinged disc, slightly yellowish context, dark yellow to ocher spore print, 
and pileipellis with septate pileocystidia. The morphological characteristics are illustrated in 
detail and compared with those of similar species. Identification of R. jilinensis was supported 
by the molecular phylogenetic analysis based on the ribosomal DNA internal transcribed 
spacer regions (ITS). 


Key worps —Russulales, taxonomy, morphology, Basidiomycota 


Introduction 

Northeast China, including Liaoning, Jilin, and Heilongjiang Provinces 
and the eastern part of Inner Mongolia Autonomous Region, covers an area 
of 1.236x10° km? (39°-53°30' N 115°-135°E) within the temperate to boreal 
continental climate zones. Plant communities range from grassland (eastern 
Inner Mongolia) to broadleaf forest (southern Liaoning), while the three main 
mountain systems (Great Hinggan, Lesser Hinggan, Changbai) are mostly 
covered by coniferous or mixed coniferous—broadleaf forests. The main trees 
in northeast China are Pinus pumila, P koraiensis, Larix gmelinii, Betula 
platyphylla, Abies nephrolepis, Picea jezoensis, and Quercus mongolica (Jiang et 
al. 2003, Xu et al. 2008). In summer and early autumn, these ectomycorrhizal 
commensal plants facilitate the fruiting of ectomycorrhizal basidiomycetes, 
among which the members of Russula Pers. are very common. 

Russula is widely distributed from western Europe to North America in the 
northern hemisphere (Romagnesi 1967, Singer 1986, Sarnari 1998, Miller & 
Buyck 2002, Bau et al. 2008). Although some new species and varieties have 
been reported from southern and southwestern China (Singer 1935, Chiu 1945, 
Ying 1983, 1989, Bi & Li 1986, Wen & Ying 2001, Wang et al. 2009), Russula 


50 ... Li&al. 


species have not been studied systematically elsewhere in China, including the 
north. Because of its taxonomic difficulty and the insufficiency of intensive 
taxonomic studies in China (Wang et al. 2009), many Russula species reported 
from northeastern China are identified using European or American names 
and lack voucher support (e.g., Teng 1996, Xie et al. 1986, Li & Bau 2003, Bau 
2004). As now in North America (Adamcik & Buyck 2010, 2011; Buyck & 
Hofstetter 2011), the lack of endemic systematic studies has probably led to 
incorrect estimates of the extent of Russula diversity in northeastern China,. 

Although many wood-inhabiting fungi were recently reported from 
northeastern China (Dai & Penttila 2006, Yuan et al. 2006, Wei & Dai 2007, Dai 
et al. 2008, Xiong et al. 2008, Dai 2010), only a few Russula species have been 
reported from this area (Song et al. 2007). In addition, many virgin and natural 
forests in this area have not yet been intensively investigated. During a survey 
on Russulales in the Changbai Mountains, one interesting Russula was found, 
which is described here as a new species. 


Materials & methods 

Specimens were collected from the Changbai Mountains, Antu County of Jilin 
Province, from 2008 to 2010. Macromorphological characteristics of the fresh fruiting 
bodies were recorded in field notes. Color names and codes follow Ridgway (1912) 
and spore print colors were categorized according to Romagnesi (1967). Collections 
were oven-dried at 50-60 °C until their water content was <15% prior to microscopic 
examination. Solutions of 10% FeSO, and sulfovanillin (SV) were used for testing the 
chemical reaction of dried specimens. Specimens for microscopic examinations were 
hand-sectioned and rehydrated in a 5% KOH solution prior to observation under a 
Nikon Eclipse 80i microscope. Basidiospores (examined in Melzer’s reagent), basidia, 
pleurocystidia, cheilocystidia and elements of the pileipellis and stipitipellis were 
measured, with at least 20 elements measured for each character. The abbreviation 
(n/m/p) = n spores from m basidiospores of p specimens. Basidiospore dimensions 
are shown as (a—) b-c (-d), with the range b-c including 90% of the measured values 
and the extreme values shown in parentheses. The spore quotient (Q) = spore length 
divided by spore width. Q (in bold) = average quotient value + standard deviation. Spore 
ornamentations and apiculi were not included in measurements. Further explanations 
of basidiospore data follow Yang (2000). Scanning electron images were also captured 
with an FEI Quanta 200 electron microscope. Abbreviations of herbarium names follow 
Holmgren et al. (1990). Specimens cited were deposited in the Mycological Herbarium, 
Institute of Microbiology, Chinese Academy of Sciences (HMAS). 

For phylogenetic studies, genomic DNA was extracted from dried fruiting bodies 
briefly crushed with quartz sand in liquid nitrogen. DNA template for amplification 
was prepared using a BioTeke plant DNA rapid extraction kit. PCR was conducted with 
an Eastwin Dragon Black Vajra thermo cycler using ITS1 (5’-GTAGGTGAACCTGCGG- 
3°) and ITS4 (5’-rccTrccGCTTATTGATATGC-3’) primers (Shanghai Sangon, Beijing 
TransGen Biotech), EasyTaq DNA polymerase, dNTPs, and PCR buffer to amplify 
the ITS1+5.8S+ITS2 region. Amplification of a 50 uL mixture — 35.5 ul ddH,O, 5 ul 


Russula jilinensis sp. nov. (China) ... 51 


PCR buffer, 4 ul dNTP (2.5 mmol/L), 1 ul DNA template, 2 ul (10 umol/L) each of 
two primers, 0.5 ul (Su/ul) Taq DNA polymerase — followed a protocol of 5 min at 
94°C (initial denaturation), 35 cycles of 1-min at 94°C (denaturation) + 1 min at 56°C 
(annealing) + 1 min at 72°C (extension), and a final 10-min extension at 72°C. The PCR 
products were electrophoresed in ethidium bromide-stained agarose gel and sequenced 
by SinoGenoMax Co. Ltd, using an ABI 3730XL Analyzer and ABI BigDye 3.1 Cycle 
Sequencing Kit. The sequences were deposited in GenBank. 

Sequences were aligned and edited in ClustalX (Thompson et al. 1997) and Bioedit 
(Hall 1999). Phylogenetic analysis was performed with MEGA version 4.1 Beta (Tamura 
et al. 2007), and Minimum Parsimony trees were constructed with the test of inferred 
phylogeny a bootstrap of 1000 replications and a random seed (Eck & Dayhoff 1966, 
Rzhetsky & Nei 1992). The MP tree was obtained using the Close-Neighbor-Interchange 
algorithm with search level in which the initial trees were obtained with the random 
addition of sequences (10 replicates). The tree was drawn to scale, with branch lengths 
calculated using the average pathway method and in the units of the number of changes 
over the whole sequence (Nei & Kumar 2000). All positions containing gaps and missing 
data were eliminated from the data set (Complete Deletion option). The bootstrap 
consensus tree inferred from 1000 replicates is taken to represent the evolutionary 
history of the taxa analyzed. Branches corresponding to partitions reproduced in less 
than 50% bootstrap replicates are collapsed. Of the 424 positions in the final dataset, 
253 are parsimony informative. The ITS1-5.8S-ITS2 sequences of Albatrellus ovinus 
(Schaeff.) Kotl. & Pouzar and Bondarzewia mesenterica (Schaeft.) Kreisel (as B. montana) 
were used as outgroups (Wang et al. 2009). 


Taxonomy 


Russula jilinensis GJ. Li & H.A. Wen, sp. nov. Fics 1-4 
MycoBank MB 563682 

Differs from Russula integra by shorter basidia and pleurocystidia and smaller spores. 

EryMo_Locy: named after the type locality. 

Type: CHINA, Jilin Province, Antu County, Erdaobaihe, Changbai Mountains, 42°24'N 

128°04'E, alt. 761 m, on ground in mixed coniferous—broadleaf forest dominated by 

Pinus koraiensis and Picea, 1 August 2008, M.X. Zhou & Y. Gafforov 08004 (Holotype, 

HMAS194253, GenBank GU966632). 
PiLEus 6.1-7.3 cm broad, first hemispheric, convex, then more or less convex 
to plano-convex, depressed weakly in the center when mature, colors Jasper 
Red to Coral Red (x1113'—5’) when juvenile, Pompeian Red to Dragon’s-blood 
Red (x1113'/i-5'1) with age, Nopal Red to Brazil Red (13i-5i) when dried; center 
Vinaceous-Cinnamon to Pinkish Cinnamon (xx1x13''b—15"'b) when juvenile, 
Orange-Cinnamon to Cinnamon (xx1x13'’-15') with age, Mikado Brown 
(xx1x13"i) when dried, Nopal Red (13i) in SV, color unchanged in FeSO; viscid 
when wet, glabrous, smooth, non-pruinose when dried, pellis peeling 1/2 to 
the disc; margin obtuse, not or very slightly striate when old, never cracked. 
CONTEXT 1.0-1.5 cm thick from stipe top to pileus center, brittle, white (Lu), 
Naples Yellow (xv119'd) when old or dried, color unchanged when bruised; 


52 ... Li & al. 


Fic.1: Russula jilinensis (Holotype HMAS 194253). 


Fic.2: Russula jilinensis (HMAS 262395). Scale bar = 1 cm. 


Russula jilinensis sp. nov. (China) ... 53 


4/26/2010 WD Mag HV Det Spot 
2:42:07 PM.9.6 mm 5000x 12.5 kV ETD 3.0 


Fic. 3: Russula jilinensis (Holotype HMAS194253). 
Scanning electron microscope (SEM) photo. Basidiospores. Scale bar = 10 um. 


taste mild; odor none or indistinct. LAMELLAE 4-7 mm broad, 9-12/cm at 
the edge, no lamellulae, decurrent to more or less adnate, not forked, fragile, 
Pale Yellow-Orange (11115f) first, Pale Orange-Yellow (11117f) when mature or 
dry, color unchanged in FeSO, STIPE clavate, enlarged towards the base, 9.2 x 
1.8-2.1 cm, smooth, solid but irregularly hollowing when old, white (L111) when 
juvenile, becoming Baryta Yellow, Wax Yellow, or Primuline Yellow (1v21f, 
Xv121’, xv119', respectively) towards the base when old or dry, Brazil Red (15i) 
in SV, no color change in FeSO,. SPORE PRINT dark yellow, ocher, IIc. 
BASIDIOSPORES (80/3/3) (6—)7-8(-8.5) x (5.5-)6-7 um, average 7.5 x 6.3 
um, [Q= (1.02) 1.06-1.28 (1.31), Q= 1.18 + 0.06], subglobose to ellipsoid, 
without oil droplet, plage indistinct and amyloid; ornamentation amyloid, 
mostly isolated, rarely linked by fine lines and not forming a mesh, warts 
conic to cylindrical, <0.4-1.0 um tall. Basrp1a (33-)38-44 x 11-13(-14) um, 
clavate to subclavate, broadly tapered towards the base, 4-spored, sometimes 
2-spored, hyaline in KOH, occasionally containing a large droplet; mature 
basidia projecting <15-10 um above the subhymenium; sterigmata 3-5 um. 
PLEUROCYSTIDIA (50-)53-56(-63) x (8-)9-10(-11) um, rare, emergent, 
fusiform, clavate to subclavate, often with a subacute tip, sometimes witha frayed 
small appendage and dense crystal inclusions, projecting <35 um above the 
subhymenium, color unchanged in FeSO, dark grey in SV reaction dark grey. 


54 ... Li & al. 


Fic. 4: Russula jilinensis (Holotype HMAS 194253). 
A. Pleurocystidia; B. basidia; C. terminal elements of pileipellis; D. pileocystidia. 
Scale bars = 10 um. 


CHEILOCYSTIDIA mostly similar to pleurocystidia. PILEIPELLIS two-layered, 
100-125 um thick, gelatinous; epicutis an ixotrichoderm, epicuticular hyphae 
3-6 um broad, septate, mostly unbranched, erect to suberect, tangled, hyaline 


Russula jilinensis sp. nov. (China) ... 55 


in KOH, terminal cells thin-walled, cylindrical, with obtuse, undifferentiated, 
often tapered ends; pileocystidia 48-64 x 5-7 um, dispersed, numerous in 
surface, scattered, clavate, subclavate to cylindrical, with 0-2 septa, contents 
crystalline, granulate, slightly turning grey in SV; subcutis hyphae recumbent, 
filamentous, interwoven, branched, septate, 2.5-5 um diam., hyaline to pale 
yellowish brown in KOH; trama composed of subspherical to angular 13-24 
uum diam sphaerocysts and connective hyphae. STIPITIPELLIS composed mostly 
of interwoven branched elongated 3-6 um diam hyaline hyphae with inflated 
cells; caulocystidia absent. Clamps or laticiferous hyphae absent in all tissues. 
Eco.oey: Solitary on soil in mixed coniferous—broadleaf forests, July- 
August. Known only from the Changbai Mountains. Edibility: unknown. 
ADDITIONAL SPECIMENS EXAMINED: CHINA, JILIN PROVINCE, ANTU COUNTY, Heping 
Forest Farm, 43°07'N 128°54’E, alt. 1014 m, in coniferous forest dominated by Pinus, 
22 July 2010, L.D. Guo, X. Sun, G.J. Li & L.J. Xie 20100054 (HMAS262395, GenBank 
HQ693525); Changbai Mountains Forest Ecological System Research Center, 43°23'N 


128°05’E, alt. 811 m, in mixed coniferous—broadleaf forest, 25 July 2010, X. Sun & GJ. Li 
20100410 (HMAS262364, GenBank HQ693524). 


Discussion 

Russula jilinensis is macroscopically characterized by its bright red pileus, 
light-yellow lamellae, and a white stipe without red or purple tinge. It shows 
superficial affinities with subg. Coccinula sect. Laetinae Romagn., but its 
relatively shorter basidia and larger cystidia are more characteristic of subg. 
Polychromidia sect. Integrinae Maire (e.g., R. integra f. gigas Romagn.). The R. 
jilinensis ITS1-5.8S-ITS2 sequences cluster with R. curtipes FH. Moller & Jul. 
Schaff. (Fic. 5), and a blasting with that of R. curtipes (AY061668, Miller & 
Buyck 2002) through NCBI databank shows a 93% max identification of 97% 
coverage, confirming that R. jilinensis is related to R. integra/curtipes group in 
Russula sect. Integrinae. 

Russula integra (L.) Fr., which is common in Europe, has been reported 
many times from China and neighboring East Siberia (Teng 1964, Romagnesi 
1967, Tai 1979, Xie et al. 1986, Ying et al. 1987, Knudsen & Stordal 1992, Mao & 
Zhuang 1997, Mao 2000, Bau et al. 2008). However, few specimens of R. integra 
have been recorded from northeast China. Russula integra and R. jilinensis 
are similar in spore ornamentation and cystidial shape, and both species are 
associated with Pinus and Picea in the subalpine zone (Romagnesi 1967, 1985). 
However, our microscopic observation followed by intensive comparison of 
descriptions of R. integra by Moller & Schaffer (1935) and Romagnesi (1967) 
indicates that R. jilinensis is a different species. The latter has shorter basidia 
and pleurocystidia and smaller spores than those of R. integra (with spores 
8.2-10 x 7-9.2 um, basidia 45-65 x 11-14.5 um, cystidia 82-120 x 8.5-13 um, 
in Romagnesi 1967). 


56 ... Li&al. 


R, jilinensis HQ693525 (HMA S 262395) 
— R. jilinensis GU966632 (HMA S 194253) 
R, jilinensis HQ693524 (HMA S 262364) 
93 p R.aff.curtipes EU248593 
R.curtipes AY061668 
100 p— R.laricina AY061685 
R.cessans AY061730 
R.caerulea AY061661 
96 R.roseipes AY061716 
R.tarci AY061720 
R.paludosa AY061703 
99 p R.integra AY061683 
R.integriformis AY 061684 
61 R.xerampelina AY061734 
10 R.pascua AY061705 
R.nitida AY061696 
R.sphagnophila AY061719 
R.nauseosa AY061733 
98 yR.cuprea AY061667 
R.adulterina AY061651 
98 R.clariana AY 061664 
R.violacea AY061725 
99 pR.lepida AY061686 
R.aurantiaca AY061658 
54 R.consobrina AY061666 
R.gracillima AY061678 
R.persicina AY061707 
R.queletii AYO61711 
99 p= R.drimeia AY061672 
R.sanguinea AY061718 
96 p= R. bicolor GU966633 
st bad R. bicolor FJ845435 
R.raoultii AF418621 
R.aquosa AY061657 
oe R.raoultii AY061712 
9g [7 R-nana AY061694 
TA, R.emetica AY061673 
R.betularum AY061729 
R.romellii AY0617 14 
R.aurea AY061659 
100 A.ovinus AY 293165 
B.montana AF042646 


20 


Fic. 5. One of 13 ITS1-5.8S-ITS2-based phylogenies of Russula jilinensis and related Russula species, 
using the Maximum Parsimony method. The percentages of replicate trees in which the associated 
taxa clustered together in the bootstrap test (1000 replicates) are shown above the branches. 


Another phylogenetically related species is R. curtipes, which shares with 
R. jilinensis the short basidia and firm and yellowing context of the R. integra- 
curtipes group, but they distinctly differ from each other in pileus color and 
habitat: R. curtipes has a dark wine-red, haematite red, to light russet vinaceous 
pileus and associates with beech in western Europe with a possible preference 
for limestone (Romagnesi 1967), while R. jilinensis has a brighter red pileus and 
grows in mixed forests dominated by conifers in efflorescent basalt soils. The 
reddish pileus of R. jilinensis distinguishes it from most of the typical violet- 
or purple-capped members of R. sect. Integrinae such as R. romellii Maire and 
R. carpini R. Girard & Heinem. Another species in R. sect. Integrinae with a 
dark-red pileus, R. rubroalba (Singer) Romagn., shares a more or less similar 
pileus color and spore ornamentation but is associated with a deciduous forest 
and has much longer cystidia than those of R. jilinensis. 


Acknowledgments 
The authors are grateful to Dr. Y.C. Dai (Institute of Applied Ecology, Chinese 
Academy of Sciences) and Dr. Kanad Das (Botanical Survey of India, Sikkim Himalayan 


Russula jilinensis sp. nov. (China) ... 57 


Regional Centre) for reviewing the manuscript; to Ms. H.M. Li, Ms. A.R. Yan and Dr. 
T.Z. Wei (HMAS) for providing the herbarium specimens; to Ms. X.E. Zhu for inking 
in line drawings, to Dr. Z.L. Yang (Kunming Institute of Botany, CAS), B.A. Jaffee 
(University of California Davis), Dr. S. Pennycook (Landcare Research), Dr. L. Norvell 
(Pacific Northwest Mycology Service), and Dr. J.Y. Zhuang for improving the manuscript; 
to Dr. L.D. Guo, Dr. X. Sun, Dr. Z. Yong, Mr. M.X. Zhou, Dr. Y. Gafforov, Ms. B. Wu, 
Mr. C. Gao, Ms. G.Y. Li, Ms. X.L. Yang, Ms. J.Z. Li, Ms. Q. Qian, Mr. Z.M. Li, Ms. S.F. 
Yue, Mr. D. Zhao, Ms. J.X. Yang (Institute of Microbiology, CAS), Ms. L.J. Xie, Dr. C.Y. 
Sun and Ms. J. Zhou (National Institute of Occupational Health and Poison Control, 
China CDC) for assistance in collecting specimens; to Dr. C.L. Li and Ms. J.N. Liang 
(Institute of Microbiology, CAS) in SEM photographing. This project was supported by 
the National Natural Science Foundation of China (No. 30770013). 


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ISSN (print) 0093-4666 © 2012. Mycotaxon, Ltd. ISSN (online) 2154-8889 


MY COTAXON 


http://dx.doi.org/10.5248/120.59 
Volume 120, pp. 59-66 April-June 2012 


Acrogenospora hainanensis sp. nov. and 
new records of microfungi from southern China 


JIAN Ma, Li-Guo Ma, Y1-DONG ZHANG, JI-WEN XIA & XIU-GUO ZHANG 


Department of Plant Pathology, Shandong Agricultural University, Taian, 271018, China 
*CORRESPONDENCE TO: zhxg@sdau.edu.cn, sdau613@163.com 


ABSTRACT —Acrogenospora hainanensis is described and illustrated from specimens 
collected on dead branches in Hainan Province, China. The fungus is characterized by 
terminal, monoblastic, percurrently extending conidiogenous cells with solitary, spherical or 
subspherical, non-septate and relatively small conidia. Acrogenospora gigantospora, Nakataea 
fusispora, and Pseudospiropes costaricensis are newly recorded from China. 


KEY worps — anamorphic fungi, taxonomy 


Introduction 

The forests of southern China have a rich mycota, and mycological 
investigations dealing with many new species have been published recently 
(Dai & Cui 2006, Cui & Dai 2008, Cui et al. 2009a,b, 2011, Zhang et al. 2009, 
2011, Ma et al. 2010). During ongoing mycological surveys in the forests of 
Hainan and Guangdong provinces, four interesting anamorphic fungi clearly 
related to Acrogenospora, Nakataea, and Pseudospiropes were collected on dead 
branches. One of these is an undescribed species of Acrogenospora, while the 
other three species are new records for China. 


Acrogenospora hainanensis Jian Ma & X.G. Zhang, sp. nov. Fic. 1 
MycoBank MB 564117 


Differs from Acrogenospora verrucispora in smaller smooth conidia and from A. 
sphaerocephala in smaller conidia and more narrow conidial scars. 


Type: China, Hainan Province: Tunchang, on dead branches of unidentified broad- 
leaved tree, 12 April 2011, J. Ma (Holotype HSAUP H5509; isotype HMAS 146167). 


EryMo_oey: refers to the province where the type was found. 


Colonies on the natural substratum effuse, black, glistening, hairy. Mycelium 
mostly immersed, composed of branched, septate, subhyaline to pale brown, 


60 ... Ma & al. 
D E 
10m 


Fic. 1. Acrogenospora hainanensis (holotype). A-C. Conidiophores, conidiogenous cells 
with conidia. Note that in C the mature conidium is pushed sidewise following successive 
percurrent extension of the conidiogenous cells. D. Conidiophores. E. Conidia. 


mt 


uioT 
ungoT 


smooth-walled hyphae. Conidiophores macronematous, mononematous, 
single, unbranched, erect, straight or slightly flexuous, septate, smooth, 
brown to dark brown, paler towards the apex, 60-80 um long, 2-3.5 um thick. 
Conidiogenous cells monoblastic, integrated, terminal, cylindrical, smooth, 
pale brown, proliferating percurrently. Conidial secession schizolytic. Conidia 
solitary, acrogenous, dry, spherical or subspherical, smooth, brown, non- 
septate, 7.5-9.5 x 7-8.5 um, with a truncate base 1.5-2 um wide. 


COMMENTS — Acrogenospora, with Monotospora sphaerocephala Berk. & 
Broome [= A. sphaerocephala (Berk. & Broome) M.B. Ellis] as the type 
species, is characterized by integrated, terminal, monoblastic, percurrently 
extending conidiogenous cells on distinct, unbranched conidiophores, and 
acrogenous, solitary, non-septate conidia with schizolytic conidial secession. 
These characters separate the genus from Domingoella Petr. & Cif. (Petrak 
& Ciferri 1932), Brachysporiella Bat. (Batista 1952), and Endophragmiella B. 
Sutton (Sutton 1973). Ten species are currently included in Acrogenospora, all 
discovered on dead branches, rotten wood, or submerged bamboo (Ellis 1971, 
1972, Hughes 1978, Goh et al. 1998, Zhu et al. 2005, Hu et al. 2010). The species 
are distinguished primarily on conidial shape, size, and ornamentation (Hughes 
1978, Goh et al. 1998, Zhu et al. 2005). 


Acrogenospora hainanensis sp. nov. (China) ... 61 


Of the known species, Acrogenospora hainanensis is most similar to 
A. sphaerocephala (Ellis 1971) and A. verrucispora Hong Zhu et al. (Zhu et al. 
2005) in the production of spherical or subspherical conidia. However, the new 
species differs from A. verrucispora (verrucose conidia 15-33 x 14-33 um) in 
its smaller smooth-walled conidia and from A. sphaerocephala (19-21.5 um 
diam., conidial scars 5-7 um wide) in its smaller conidia and narrower conidial 
scars. 


Acrogenospora gigantospora S. Hughes, New Zealand J. Bot. 16: 314, 1978. Fic. 2 

Colonies on the natural substratum effuse, black, glistening, hairy. Mycelium 
mostly immersed, composed of branched, septate, pale brown, smooth-walled 
hyphae. Conidiophores distinct, single or in groups of 2-4, unbranched, erect, 
straight or slightly flexuous, septate, smooth, brown to dark brown, up to 265 um 
long, 5.5-8.5 um thick. Conidiogenous cells monoblastic, integrated, terminal, 
cylindrical, smooth, pale brown, with multiple percurrent extensions. Conidial 
secession schizolytic. Conidia solitary, acrogenous, dry, broadly obovoid to 


Fic. 2. Acrogenospora gigantospora. A-C. Conidiophores, conidiogenous cells with 
conidia. Note that in A and B the mature conidium is pushed sidewise following successive 
percurrent extension of the conidiogenous cells. D. Conidia. 


A B C 


ind 
— 
E 


62 ... Ma & al. 


subspherical, smooth, dark brown to black, non-septate, 37-42 x 24-30 um, 
with a truncate base 5-7.5 um wide. 
SPECIMEN EXAMINED: CHINA, GUANGDONG PROVINCE: Chebaling National Nature 
Reserve, on dead branches of unidentified broad-leaved tree, 19 Oct 2010, J. Ma (HSAUP 
H5402). 
COMMENTS - Acrogenospora gigantospora is reported for the first time from 
China. Except for its shorter narrower conidiophores, our collection fits well 
with the original description of A. gigantospora (Hughes 1978; conidiophores 
< 700 x 9-14.5 um). Acrogenospora gigantospora appears to be most closely 
related to A. ovalis Goh et al. (Goh et al. 1998) in conidial shape but differs 
in having larger conidia and a wider conidial scar. In addition, mature 
A. gigantospora conidia are brown to dark brown while those of A. ovalis are 
mid orange-brown. 


Nakataea fusispora (Matsush.) Matsush., Icon. Microfung. Matsush. Lect.: 100, 

1975. FIG. 3 

= Vakrabeeja fusispora Matsush., Microfungi Solomon Is. Papua-New Guin.: 66. 1971. 

= Pyricularia fusispora (Matsush.) Zucconi, Onofri & Persiani, Micol. Ital. 13(2): 9. 1984. 

Colonies on the natural substratum effuse, dark brown, hairy. Mycelium 
partly superficial, partly immersed in the substratum, composed of branched, 
septate, smooth-walled hyphae. Conidiophores conspicuous, mononematous, 
unbranched, straight or flexuous, smooth, septate, brown, paler towards the apex, 
64-95 um long, 3.5-4.5 um thick. Conidiogenous cells polyblastic, integrated, 
terminal becoming intercalary, sympodially proliferating, denticulate, thin- 
walled; denticles subcylindrical, flat and open at the apex, septate at the base. 
Conidia solitary, dry, apical and lateral, verruculose, fusiform, acute at the apex, 
with a denticle at the base, 3-septate, the cell at each end hyaline or very pale 
brown, central cells pale to mid brown; conidia 22-34 x 6-7.5 um. 
SPECIMEN EXAMINED: CHINA, HAINAN PROVINCE: tropical forest of Bawangling, on 
dead branches of unidentified broad-leaved tree, 11 Dec 2009, J. Ma (HSAUP H5125). 

ComMENtTs — Nakataea, established by Hara (1939) with Helminthosporium 
sigmoideum Cavara [= N. sigmoidea (Cavara) Hara] as the type species, is 
characterized by solitary, apical and lateral conidia seceding rhexolytically 
from polyblastic, integrated, terminal becoming intercalary, sympodially 
extending conidiogenous cells. The conidia are falcate, sigmoid, fusiform 
or obclavate, almost always 3-euseptate, versicolored, end cells paler than 
the central cells, with a distinct basal frill (Ellis 1971). Vakrabeeja Subram. 
(Subramanian 1956) is based on the same type species as Nakataea and therefore 
a superfluous homotypic synonym. Kirk (1983) suggested that Nakataea 
should be synonymised with Pyricularia Sacc., because their type species were 
morphologically similar and both had Magnaporthe teleomorphs. ITS rRNA 


Acrogenospora hainanensis sp. nov. (China) ... 63 


D, E 


UINQZ 


Fig. 3. Nakataea fusispora. A-C. Conidiophores, conidiogenous cells with denticles and 
conidia. D. Conidiophore and conidiogenous cell. E. Conidia. 


sequence phylogenetic analyses (Bussaban et al. 2005) suggest that N. fusispora 
is distinct from Pyricularia species and should be maintained in Nakataea. 

Nakataea fusispora is most similar to N. sigmoidea (Hara 1939) in conidial 
shape but can be separated from N. sigmoidea (conidia 40-83 x 11-14 
um, smooth) by its smaller conidia with verruculose walls. The conidia of 
our collection are somewhat wider than cited in the original description 
by Matsushima (1971; 4.8-6 um). The conidial length ranges of our and 
Matsushima’s material overlap, and other morphological features of our fungus 
satisfy the original description of N. fusispora. This fungus is reported for the 
first time from China. 


Pseudospiropes costaricensis (E.F. Morris) de Hoog & Arx, Kavaka 1:59, 1974 
19737] FIG. 4 
= Pleurophragmium costaricensis E.F. Morris, Mycologia 64: 893. 1972. 

Colonies on natural substratum effuse, brown, hairy. Mycelium partly 
superficial, partly immersed in the substratum, composed of branched, 
septate, smooth-walled hyphae. Conidiophores distinct, single or in groups, 
erect, unbranched, straight or flexuous, smooth, septate, brown to dark brown, 
paler toward the apex, 160-290 um long, 6-9 um wide. Conidiogenous cells 
holoblastic, polyblastic, indeterminate, terminal becoming intercalary, pale 
brown to brown, integrated, always with holoblastic sympodial extensions. 


64 ... Ma & al. 


Fic. 4. Pseudospiropes costaricensis. A-B. Conidiophores and conidiogenous cells with 
conidia. C. Conidiophores and conidiogenous cells. D. Conidia. 


Conidiogenous loci enlarged, thickened, protuberant and black, lenticular. 
Conidial secession schizolytic. Conidia apical and lateral, solitary, dry, simple, 
obclavate or fusiform, pale brown or brown, smooth, 8-11-distoseptate, 40-53 
x 11-14 um, apical cell with a hyaline vesicle, basal cell with a truncate base, 
2-3.5 um. 

SPECIMEN EXAMINED: CHINA, GUANGDONG PROVINCE: Chebaling National Nature 

Reserve, on dead branches of unidentified broad-leaved tree, 19 Oct 2010, J. Ma (HSAUP 

H5419). 
CoMMENTS - Since the establishment of Pseudospiropes M.B. Ellis, based on 
Helminthosporium nodosum Wallr. [= P. nodosus (Wallr.) M.B. Ellis], there have 
been numerous additions to the genus. However, Sinclair et al. (1997) pointed 
out that Pseudospiropes is heterogeneous. Castafieda et al. (2001) reviewed the 
genus and proposed three new segregate anamorphic genera —Minimelanolocus 
R.F. Castafieda & Heredia, Nigrolentilocus R.F.Castafieda & Heredia, and 
Matsushimiella R.F.Castafieda & Heredia— based on the morphology of 
conidiogenous loci, conidial septation, and conidial secession. As presently 
constituted, Pseudospiropes is characterized by polyblastic, integrated, terminal 
becoming intercalary, sympodial, cicatrized conidiogenous cells bearing 
broadly enlarged, thickened, often dark and protuberant scars, and solitary, 
apical and lateral, distoseptate conidia with schizolytic conidial secession (Ellis 
1971, Castaneda et al. 2001). Currently, Pseudospiropes contains 13 valid species 
(Ma et al. 2011). 


Acrogenospora hainanensis sp. nov. (China) ... 65 


Pseudospiropes costaricensis was originally assigned to Pleurophragmium 
Costantin and subsequently transferred to Pseudospiropes by de Hoog & Arx 
(1974) due to its cicatrized conidiogenous cells with large, often dark and 
prominent scars. The conidia of Pseudospiropes costaricensis are morphologically 
similar to those of P. hachijoensis Matsush. (Matsushima 1975), P josserandii 
(Bertault) Iturr. (Iturriaga & Korf 1990), P nodosus, and P. simplex (Nees & 
T. Nees) M.B. Ellis (Ellis 1971). However, P. costaricensis can be easily separated 
from P. hachijoensis, P. josserandii, P. nodosus and P. simplex by its conidia, which 
have a hyaline vesicle at the apex. Except for having more conidial septa, our 
collection overlaps well with the original description of P. costaricensis (Morris 
1972; 6-9 septa). This fungus is reported as new to the Chinese mycobiota. 


Acknowledgments 

The authors express gratitude to Dr Bryce Kendrick and Dr Rafael F. Castafieda-Ruiz 
for serving as pre-submission reviewers and for their valuable comments and suggestions. 
This project was supported by the National Natural Science Foundation of China (Nos. 
31093440, 30499340, 30770015) and the Ministry of Science and Technology of the 
People’s Republic of China (Nos. 2006FY120100, 2006FY110500-5). 


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ISSN (print) 0093-4666 © 2012. Mycotaxon, Ltd. ISSN (online) 2154-8889 


MY COTAXON 


http://dx.doi.org/10.5248/120.67 
Volume 120, pp. 67-73 April-June 2012 


A new species of Nectria on Populus from China 


ZHAO-QING ZENG*” & WEN- YING ZHUANG" 


' State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 
100101, China 

? Graduate University of Chinese Academy of Sciences, Beijing 100049, China 

CORRESPONDENCE TO *: zhuangwy@im.ac.cn 


ABSTRACT - A new species, Nectria zangii, is described on Populus branches from 
Donglingshan in western Beijing. The anatomy of perithecia, which become cupulate when 
dry, and positive reactions to KOH and lactic acid of the fungus indicates that this species 
belongs in the genus Nectria. It is characterized by small, non-septate, allantoid ascospores 
and small, subcylindrical to narrowly clavate asci. Sequence analysis of the combined nuclear 
ribosomal DNA ITS1-5.8S-ITS2 and partial 6-tubulin gene confirm its taxonomic position 
in Nectria as a species new to science. 


KEY worps - taxonomy, morphology 


Introduction 

The genus Nectria (Fr.) Fr. was established in 1849 and typified by 
N. cinnabarina (Clements & Shear 1931). About 34 species are currently 
accepted in the genus (Rossman et al. 1999, Kirk et al. 2001, Dobbeler 2005a,b, 
Marincowitz et al. 2008, Pande 2008). Ten species are known from China (Teng 
1963, Tai 1979, Wang et al. 1999, Zhuang & Zhang 2002, Zhang & Zhuang 
2003a,b). The genus is characterized by well-developed stromata, subglobose, 
globose to ellipsoid, red to dark red, fleshy, soft-textured, uniloculate, warted 
perithecia that become cupulate when dry, and associated with coelomycetous 
anamorphs (Rossman et al. 1999, Hirooka et al. 2009). Ascospores of Nectria 
are variable and usually broadly ellipsoid to long-fusiform, hyaline to yellow- 
brown, smooth to striate, non- to multi-septate or muriform. Members of the 
genus are typically weak parasites of woody plants, and occur on hardwood 
trees and shrubs throughout temperate regions of the Northern Hemisphere 
(Samuels et al. 2009, Hirooka et al. 2011). 

In this study, a new species of Nectria with small allantoid non-septate 
ascospores and subcylindrical narrow asci is described. On the basis of 
morphology and sequence analysis of nuclear ribosomal DNA ITS1-5.8S-ITS2 


68 ... Zeng & Zhuang 


(ITS) and partial B-tubulin gene, its position in Nectria is confirmed, and its 
relationship with similar species of the genus is discussed. 


Materials & methods 

The taxonomic treatments and methods described in Luo & Zhuang (2010) were 
generally followed for morphological studies. Water was used as the mounting medium 
for microscopic examinations and measurements. Photographs were taken from water 
or lactophenol cotton blue mounts with a Canon G5 (Tokyo, Japan) digital camera 
connected to a Zeiss Axioskop 2 plus microscope (Géttingen, Germany). For anatomic 
studies, longitudinal sections through ascomata were made with a freezing microtome 
(YD-1508-II, Yidi Medical Appliance Factory, Jinhua, Zhejiang) at a thickness of ca. 
8 um. Continuous measurements of individual structures are based on 30 measurements, 
except when otherwise noted. Single-spore isolates were obtained from recent collections. 
Colony characters were recorded from cultures on potato dextrose agar (PDA, Gams et 
al. 1998). Microscopic descriptions of the anamorphs were taken from cultures after 
14 days. Specimens examined are deposited in the Mycological Herbarium, Institute of 
Microbiology, Chinese Academy of Sciences (HMAS), and cultures are kept in the State 
Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences. 

DNA was extracted from mycelium harvested from colonies on PDA after 2 weeks 
(Wang & Zhuang 2004). Sequences of ITS and the partial 6-tubulin gene were amplified 
with primer pairs ITS5-ITS4 (White et al. 1990) and T1-T222 (O'Donnell & Cigelnik 
1997) respectively. The PCR reaction mixture consisted of 2.5 ul 10x PCR buffer, 1.5 ul 
MgCl, (25 mM), 1.25 pl each primer (10 uM), 0.5 pl dNTP (10 mM each), 1.25 ul DNA 


TABLE 1. Materials used in this study. 


SPECIES COLLECTION NO. Hoan DNS Paulin 
GENBANK NO. GENBANK NO. 
Cosmospora coccinea Rabenh. CBS 114050 HM484537 HM484589 
Nectria australiensis Seifert HMAS 83397 GU075855 HM054111 
N. berolinensis (Sacc.) Cooke CBS 126112 HM484543 HM484594 
N. cinnabarina (Tode) Fr. CBS 125150 HM484684 HM484820 
CBS 125158 HM48 4696 HM484830 
N. coryli Fuckel CBS 129156 HM484539 HM484596 
N. miltina (Mont.) Mont. CBS 121121 HM484547 HM484609 
N. nigrescens Cooke CBS 125148 HM484707 HM484806 
CBS 129360 HM484711 HM484808 
N. pseudocinnabarina Rossman CBS 128673 HM484553 HM484608 
N. pseudotrichia (Schwein.) HMAS 183559 HMo54154 HMo54115 
Berk. & M.A. Curtis HMAS 183175 HMo054138 HMo54114 
N. zangii HMAS 251258 JN9974248 JN997421 
holotype, HMAS 251247 JN997425 JN997422 
HMAS 188502 JN997426 JN997423 
Neonectria ramulariae Wollenw. CBS 151.29 HM054150 HM054124 


“Numbers in bold = newly submitted sequences. 


Nectria zangii sp. nov. (China) ... 69 


template, 0.25 ul Taq polymerase (5 U/ul) and 16.5 ul ddH,O. Reactions were performed 
on an ABI 2720 Thermal Cycler (Gene Co. Ltd.) with cycling conditions of denaturation 
at 95°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 52°C 
(ITS region) and at 55°C (B-tubulin gene) for 30 s and elongation at 72°C for 45 s, with 
a final extension step at 72°C for 10 min to complete the reactions. PCR products were 
purified with the PCR Product Purification Kit (Biocolor BioScience & Technology Co.) 
and sequenced with the ABI BigDye Terminator V3.1 Cycle Sequencing Kit (Applied 
Biosystems) on an ABI 3730 XL DNA Sequencer (SinoGenoMax Co. Ltd.). ITS5 and 
ITS4 (for ITS), and T1 and Bt2b (for the partial B-tubulin gene) were employed as 
sequencing primers (White et al. 1990, Glass & Donaldson 1995, O’Donnell & Cigelnik 
1997). Sequences of related species were retrieved from GenBank and are listed in 
TABLE l. 

All sequences were aligned using ClustalX 1.8 (Thompson et al. 1997), and the 
alignments were visually adjusted by BioEdit 7.0.5 (Hall 1999) when necessary. A 
partition homogeneity test was performed with 1000 replicates in PAUP 4.0b10 
(Swofford 2002) to evaluate statistical congruence between sequence data from ITS and 
B-tubulin gene regions. The partition homogeneity test (P = 0.01) suggested that the 
individual partitions were congruent (Farris et al. 1995, Cunningham 1997). A neighbor- 
joining tree was generated using MEGA 4.10 (Tamura et al. 2007) based on combined 
sequences of ITS and partial B-tubulin gene with Cosmospora coccinea and Neonectria 
ramulariae as the outgroup taxa. The Kimura 2-parameter was selected as the nucleotide 
substitution model, and gaps or missing data were pairwise deleted. Bootstrap analyses 
were performed with 1000 replicates to test phylogeny branch support. 


Taxonomy 


Nectria zangii Z.Q. Zeng & W.Y. Zhuang, sp. nov. FIGuRE 1 
MYCOBANK 563722 
Differs from Nectria miltina by thinner perithecial walls, narrower asci, smaller 
ascospores, and occurrence on twigs of Populus sp. 
Type: China. Beijing, Donglingshan, alt. 1150 m, on branches of Populus sp., 20 July 
2011, Z.Q. Zeng & H.D. Zheng 7684 (Holotype, HMAS 251247). 


Erymo.oey: The specific epithet honors the late Chinese mycologist, Prof. M. Zang. 


Ascomata perithecial, gregarious, up to 40 in a group, with a well-developed 
stroma that is erumpent through bark, superficial, subglobose to globose, 
168-200 um high, 151-203 um diam., becoming cupulate upon drying, red 
when fresh and reddish brown when dry, turning dark red in 3% KOH aqueous 
solution and orange-red to orange in lactic acid, surface somewhat roughened 
when dry; with warts or a irregular covering layer of various thickness, 0-32 
uum high, cells angular, 3.5-15 x 2.5-5 um, wall 1-1.5 um thick. Ascomatal wall 
16-26 um thick, of two layers; outer layer 10-16.5 um thick, cells angular, 2-7 
x 3-6 um, wall 1-1.5 um thick; inner layer 5-10.5 um thick, cells flattened, 
9.5-15.5 x 1.5-3.5 um, wall 1-1.5 um thick. Asci subcylindrical to narrowly 
clavate, 8-spored, with an apical ring, 28-35 x 2.5-3 um (n = 50), biseriate 


70 ... Zeng & Zhuang 


100 micron 20 micron 


PDA, obverse. c. Colony on PDA, reverse. d. Median section of ascomata. e. Structure of ascoma at 
apical portion. f. Asci with ascospores. g. Conidiophores and conidia. 


to irregularly biseriate. Ascospores allantoid to rod-shaped, straight to slightly 
curved, non-septate, hyaline, smooth, 3.5-5.5(-6) x 0.9-1.2(-1.4) um (n = 
50). 

In culture, colonies reaching 57 mm in diameter after 7 days on PDA at 25°C 
under daylight, aerial mycelium sparse to absent, yellowish, submerged mycelia 
forming a root-like or vein-like structure, reverse light orange. Conidiophores 
unbranched to sparsely branched, septate, 29-79 x 2-3.2 um, arising from agar 
surface throughout the colony, dominating near the margin. Microconidia 
allantoid to rod-shaped, not or slightly curved, hyaline, aseptate, 3-6 x 0.8-1 um 
(n = 50). Macroconidia and chlamydospores not observed. 

ADDITIONAL SPECIMENS EXAMINED: CHINA. BeyinG, Donglingshan, alt. 1150m, 
on rotten branch of Populus sp., 20 July 2011, Z.Q. Zeng & H.D. Zheng 7648-7651 


(HMAS 188501-188504); 7652 (HMAS 251248); 7653-7654 (HMAS 188505-188506); 
7655 (HMAS 251249); 7656 (HMAS 188507); 7657-7660 (HMAS 251250-251253); 


Nectria zangii sp. nov. (China) ... 71 


7663 (HMAS 188508); 7665-7667 (HMAS 188509-188511); 7668 (HMAS 251254); 
7669-7671 (HMAS 188512-188514); 7672 (HMAS 251255); 7673-7680 (HMAS 
188515-188522); 7681 (HMAS 251256); 7682 (HMAS 188523); 7683 (HMAS 251257); 
7685-7694 (HMAS 188524-188533); 7695 (HMAS 251258); 7696-7698 (HMAS 
188534-188536). 
Comments - Morphologically, the perithecial anatomy, perithecia becoming 
cupulate when dry, and positive reaction to KOH and to lactic acid indicates 
the new species’ position in Nectria. Among the existing species of the genus, 
N. zangii is most similar to N. miltina in shape of asci and ascospores. Nectria 
miltina, however, differs in having a thicker perithecial wall (25-35 um thick), 
wider asci (28-36 x 3.5-5 um), larger ascospores (5.5-7 x 1.5-2 um), and an 
entirely different host. Nectria miltina occurs on leaves of the monocotyledonous 
plant family Agavaceae (Rossman et al. 1999) instead of on dicotyledonous 
Populus sp. 

Sequence analysis of the combined ITS and partial B-tubulin gene from 
N. zangii and eight other Nectria species with various ascospore shapes and 
septation confirms taxonomic position of the new species (Fic. 2). Our results 
support two major distantly related clades within the sampled Nectria species. 
Nectria zangii, N. berolinensis, N. miltina, N. coryli, and N. pseudocinnabarina 
group together with high bootstrap support (99%). The latter four species were 
reported having sporodochial anamorphs (Rossman et al. 1999). The other 


81, Nectria zangii HMAS 188502 
100 |LN. zangii HMAS 251247 


94 N. zangii HMAS 251258 
88 N. berolinensis CBS 126112 
N. miltina CBS 121121 
N. coryli CBS 129156 
N. pseudocinnabarina CBS 128673 

100 —N. pseudotrichia HMAS 183175 

25 N. pseudotrichia HMAS 183559 
N. australiensis HMAS 83397 

100 ,N. cinnabarina CBS 125158 


80 
99 


96 N. cinnabarina CBS 125150 
100 N. nigrescens CBS 129360 
100 |. nigrescens CBS 125148 
Cosmospora coccinea CBS 114050 
91 Neonectria ramulariae CBS 151.29 
-————_ 


0.02 


Fic. 2. Neighbor-joining tree based on combined sequences of ITS and partial B-tubulin gene 
showing the relationships among some Nectria species. 


72... Zeng & Zhuang 


sampled species, including the type species N. cinnabarina, group together 
with 96% bootstrap support (Fic. 2). These results are identical to those by 
Hirooka et al. (2011). Nectria berolinensis, shown as sister to N. zangii with 94% 
bootstrap support, differs in larger perithecia (250-300 um diam.) and 5-9- 
septate larger (16-20 x 7-8 um) muriform elliptical ascospores (Seaver 1909). 
Nectria miltina also groups with N. berolinensis and N. zangii with relatively 
high support (88%). Thus, both morphology and DNA sequence analysis 
support the recognition of N. zangii as a new species. 


Acknowledgements 

We are very grateful to Dr. A. Y. Rossman and Dr. K. A. Seifert for serving as pre- 
submission reviewers, language corrections and valuable suggestions, Dr. L.L. Norvell 
for editorial assistance and language corrections, and to Dr. H.-D. Zheng, Dr. J. Luo and 
Ms. X. Song for technical help. This work was supported by the National Natural Science 
Foundation of China (No. 31070015), and the Knowledge Innovation Program of the 
Chinese Academy of Sciences (No. KSCX2-EW-J-6). 


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ISSN (print) 0093-4666 © 2012. Mycotaxon, Ltd. ISSN (online) 2154-8889 


MY COTAXON 


http://dx.doi.org/10.5248/120.75 
Volume 120, pp. 75-79 April-June 2012 


A new species of the lichen genus Phlyctis 
from Maharashtra, India 


GAYATRI CHITALE’ & URMILA MAKHIJA” 


Agharkar Research Institute, G.G. Agarkar Road, Pune -411 004, India 
CORRESPONDENCE TO: ' gayatri4u@gmail.com & ? uv_makhija@hotmail.com 


ABSTRACT—A new species, Phlyctis communis, characterized by 8-spored asci, 7—14(—16) 
transversely septate ascospores, and salazinic and norstictic acids, is described from India. 


Key worps—Ascomycetes, Ostropales, taxonomy 


Introduction 

Although taxonomic accounts of several lichen genera from Maharashtra 
have been published and many species have been described (Chitale et al. 2008, 
2009, 2011, Chitale & Makhija 2008, Dube et al. 2005, Dube & Makhija 2008, 
2010), one locally common and abundant interesting species has remained 
unpublished. For several years a conspicuously grayish-white unidentified 
crustose lichen was known to occur all over Maharashtra, mostly during the 
monsoons in the humid high altitude regions. This has now been identified and 
is described as a new species of the genus Phlyctis. 

The subtropical to temperate lichen genus Phlyctis (Wallr.) Flot., confirmed 
by phylogenetic sequence analysis as belonging in the Ostropales (Miadlikowska 
et al. 2006), is characterized by a smooth to verrucose crustose thallus, 
protococcoid green algal photobiont, sunken or erumpent, round apothecioid 
ascomata with indistinct thalline margins, proper exciple present or absent, 
unbranched or apically furcate paraphyses, 1-8-spored asci, and ascospores 
that are colourless, transversely septate to multicelled-muriform, thin walled, 
elongate-ellipsoid, and non-halonate. 

Phlyctis species generally contain one or several of the following depsidone 
acids: stictic, constictic, norstictic, connorstictic, hypostictic, salazinic psoromic, 
neopsoromic and/or protocetraric acids (Galloway & Guzman 1988). 

The genus is widely distributed, with 12 species listed by Kirk et al. (2008) 
and five others from Great Britain and Ireland (Benfield et al. 2009), China (Ma 


76 ... Chitale & Makhija 


et al. 2010), India (Joshi et al. 2010), and Australia (Lumbsch et al. 2011) added 
since 2008. Of these only five — Phlyctis himalayensis (Nyl.) D.D. Awasthi, 
P. nepalensis Rasanen, P. polyphora Stirt., P. karnatakana S. Joshi & Upreti, 
and P. subagelaea S. Joshi & Upreti — have been reported from the Indian 
subcontinent (Awasthi 2000, Joshi et al. 2010). 

In the present study, one new species has been discovered from Maharashtra 
that represents the first record of Phlyctis from this area. 


Materials & methods 

The specimens were examined using a stereomicroscope and a light microscope. 
Sections of the thalli and apothecia were stained with Lugol’s iodine solution. All sections 
examined were mounted in LPCB (lactophenol with cotton blue). TLC protocols 
followed Culberson & Kristinsson (1970) and White & James (1985). All specimens 
were observed under UV light (365 nm). The present study is based on the material 
preserved in the lichen herbarium of Ajarekar Mycological Herbarium (AMH) and 
recent collections by the authors from Maharashtra state. 


Taxonomy 


Phlyctis communis Chitale & Makhija, sp. nov. Fries 1-4 
MycoBank MB 563474 
Similis Phlyctidae karnatakanae sed ascosporis 7-14(-16) trans-septatis et acidum 
norsticticum et salazinicum continenti differt. 
Type: India, Maharashtra, Satara District, Mahabaleshwar, 24.9.1997, U.V. Makhija, 
(Holotype, AMH 97.52). 


Erymo oey: Latin communis, referring to the plentiful or common occurrence. 


THALLUS crustose, corticolous, grayish or greenish-white, spreading over the 
bark substratum in 5-12 cm patches, cracked, areolate, matt, sometimes glossy, 
white in cracks and 90-120 um thick at the margin; epiphloeodal algal layer 
30-35 um thick, green algae single celled, more or less globose, 5-7 um diam.; 
hypothallus black. 

Ascomarta black, round, oblong, numerous, scattered all over the thallus 
and radially arranged in small 1-2(-3.5) mm diam. circles, later grouped and 
merging into each other forming patches on the bark of tree trunks, individual 
ascomata <1 mm across, immersed in the thallus; disc black, plane to concave, 
covered by a white pruina; exciple brownish, composed of loosely interwoven 
hyphae in the peripheral region, and blackish-brown at the base; epihymenium 
dark brown, 30-33 um thick, KI-; hymenium colourless to light yellow, 
72-81 um tall, KI+ blue; paraphyses simple to branched at tips; hypothecium 
brownish, 12-15 um thick; asci 8-spored, 90-105 x 18-24 um, entire ascus KI+ 
blue. Ascospores hyaline, 7-14(-16) transversely septate, very rarely with 1-2 
vertical septa, 18-33(-45) x 6-9 um. 


Phlyctis communis sp. nov. (India) ... 77 


Ficures 1-4. Phlyctis communis (holotype): 1-2, habit; 3, vertical section of the ascocarp; 
4, transseptate ascospores. Scale bars: 1-2 = 1 mm; 3-4 = 50 um. 


CHEMISTRY— thallus K+ yellow turning red, C-, KC-, P+ yellow; UV-; 
norstictic and salazinic acids present. 


SELECTED ADDITIONAL SPECIMENS EXAMINED (total specimens = 110, all in AMH)— 
INDIA. MAHARASHTRA: KOLHAPUR DISTRICT, Ajra, 6.10.2004, B.C. Behera, N. Verma, 
AMH 04.320, U.V. Makhija & G.S. Chitale, AMH 04.389, Amba, 16.10.1974, C.R. 
Kulkarni & A.V. Prabhu, AMH 74.1282, on the way to Kumbhi from Gaganbawada, 
12.10.2000, B.A. Adawadkar & K.R. Randive, AMH 00.337. Pune District, Amby 
valley, 10.2.2006, G.S. Chitale, AMH 06.181; Boma Hills, Khandala, 19.9.1974, M.B. 
Nagarkar & C.R. Kulkarni, AMH 74.691, Walwan Dam, 16.9.2002, A.V. Bhosale & G.S. 
Chitale, AMH 02.113. Satara District, Mahabaleshwar, 1.11.1973, C.R. Kulkarni, 
AMH 73.2938; Panchgani, Tata Holiday Home, 29.9.2003, U.V. Makhija & B.C. Behera, 
AMH 03.369. SInpHUDURG District, Ajra to Amboli, 7.12.1974, P.G. Patwardhan & 
A.V. Prabhu, AMH 74.2248, 10.10.2000, U.V. Makhija & V.A. Mantri, AMH 00.176. 


REMARKS— With respect to external morphology and ascospore size, Phlyctis 
communis resembles P. karnatakana, also from India, which differs in fewer 
ascospore septa (7 transsepta) and containing only norstictic acid. 


78 ... Chitale & Makhija 


Phlyctis subuncinata Stirt., also with norstictic acid and transversely septate 
ascospores, differs in having a sorediate thallus. 

Two other Phlyctis species with transversely septate ascospores — 
P. himalayensis from India and P. longifera (Nyl.) DJ. Galloway & Guzman 
from New Zealand — differ especially in having larger, 7-septate ascospores. 
Phlyctis himalayensis ascospores measure 60-75 x 6-8 um, whereas P longifera 
ascospores are 55-86 Xx 5-7 um. 

Phlyctis psoromica Elix & Kantvilas, also with transversely septate ascospores, 
has only (3-)7-septate ascospores (30-39.3-50(-52) x 4-5.2-6 um) and 
contains psoromic acid. 

The somewhat similar P polyphora from India shares a whitish thallus and 
a pruinose ascomatal disc <1 mm diam. but is distinguished by very large 
(60-110 x 7.5-9.5 um) muriform ascospores. 

Phlyctis communis is very common and has been collected from semi- 
evergreen to dry deciduous forests with relative humidity between 15-90% 
where thalli grow in huge patches on the bark of Ficus benghalensis and 
Casuarina equisetifolia where it frequently associates with Arthothelium 
awasthii Patw. & Makhija. 


Acknowledgments 

We are thankful to the Ministry of Environment and Forests, Government of India, 
New Delhi, for their financial support. The authors are grateful to G. Kantvilas and 
A. Aptroot, for reviewing the manuscript, and suggestions to improve the quality of 
the manuscript. I also thank Mr. Subhash Gaikwad, for his technical help in preparing 
illustrations. 


Literature cited 

Awasthi DD. 2000. Lichenology in Indian subcontinent. Dehra Dun (India), Bishen Singh 
Mahendra Pal Singh.1-123 p. 

Benfield B, James PW, Hitch CJB. 2009. Phlyctis. 695-696,in: CW Smith et al. (eds). The lichens of 
Great Britain & Ireland, 2nd edn. British Lichen Society, London. 

Chitale G, Makhija U. 2008. A new species of the lichen genus Brigantiaea from India. Mycotaxon 
104: 409-413. 

Chitale G, Dube A, Makhija U. 2008. The lichen genus Physcia and allied genera from Maharashtra, 
India. Geophytology 37: 13-21. 

Chitale G, Makhija U, Sharma B. 2009. New combinations and new species in the lichen genera 
Hemithecium and Pallidogramme. Mycotaxon 108: 83-92. 

Chitale G, Makhija U, Sharma B. 2011. Additional species of Graphis from Maharashtra, India. 
Mycotaxon 115: 469-480. http://dx.doi.org/10.5248/115.469 

Culberson CF, Kristinsson H. 1970. A standardized method for the identification of lichen products. 
J. Chromatogr., 46: 85-93. http://dx.doi.org/10.1016/S0021-9673(00)83967-9 

Dube A, Makhija U. 2008. A new species of Parmeliella (family Pannariaceae) from India. 
Lichenologist 40: 209-212. doi:10.1017/S0024282908007470 


Phlyctis communis sp. nov. (India) ... 79 


Dube A, Makhija U. 2010. Occurrence of four additional non-hairy species of Leptogium from 
Maharashtra, India. Lichenologist, 42(6): 701-710. 
http://dx.doi.org/10.1017/S00242829 10000332 

Dube A, Chitale C, Makhija U. 2005. The lichen genera Dirinaria and Pyxine (family Physciaceae) 
from Maharashtra, India. Phytotaxonomy 5: 83-86. 

Galloway DJ, Guzman G. 1988. A new species of Phlyctis from Chile. Lichenologist 20: 393-399. 
doi:10.1017/S0024282988000507 

Joshi S, Upreti DK, Mishra GK, Divakar PK. 2010. Two new species of the lichen genus Phlyctis in 
India. The Bryologist 113(4): 724-727. http://dx.doi.org/10.1639/0007-2745-113.4.724 

Kirk PM, Cannon PF, Minter DW, Stalpers JA. 2008. Dictionary of the fungi. 10th Edition. CABI 
Bioscience: CAB International. 771 p. 

Lumbsch HT, et al. 2011: One hundred new species of lichenized fungi: a signature of undiscovered 
global diversity. Phytotaxa 18: 1-127. 

Ma R, Li MH, Wang HY, Zhao ZT. 2010. A new species of Phlyctis (Phlyctidaceae) from China 
Mycotaxon 114: 362-366. http://dx.doi.org/10.5248/114.361 

Miadlikowska J, et al. 2006. New insights into classification and evolution of the Lecanoromycetes 
(Pezizomycotina, Ascomycota) from phylogenetic analyses of three ribosomal RNA- and two 
protein-coding genes. Mycologia 98: 1088-1103. 
http://dx.doi.org/10.3852/mycologia.98.6.1088 

White FJ, James PW 1985. A new guide to microchemical techniques for the identification of lichen 
substances. Bulletin British Lichen Society. 57(Suppl.): 1-41. 


ISSN (print) 0093-4666 © 2012. Mycotaxon, Ltd. ISSN (online) 2154-8889 


MY COTAXON 


http://dx.doi.org/10.5248/120.81 
Volume 120, pp. 81-88 April-June 2012 


Two new freshwater species of Annulatascaceae from China 


D1An-MinG Hu”, Ler Car", ALI HASSAN BAHKALIP & KEVIN D. HyDE??*#5" 


'State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, 
Beijing 100101, China 

?International Fungal Research & Development Centre, The Research Institute of Resource Insects, 
Chinese Academy of Forestry, Bailongsi, Kunming 650224, China 

*Botany and Microbiology Department, College of Science, King Saud University, 
Riyadh, Saudi Arabia 

‘School of Science, Mae Fah Luang University, Chiang Rai, Thailand 

*Institute of Excellence for Fungal Research, Mae Fah Luang University, Chiang Rai, Thailand 

* CORRESPONDENCE TO: mrcailei@gmail.com, kdhyde3@gmail.com 


ABSTRACT — Two new species belonging to Annulatascaceae are described and illustrated 
on submerged wood from a freshwater stream. Annulatascus menglensis, differentiated from 
other Annulatascus species by its hyaline ascomatal neck, is also characterized by cylindrical 
asci with a large bipartite refractive J- apical ring and hyaline fusiform ascospores without 
sheath or appendages. Aqualignicola vaginata, which is distinguished from A. hyalina in 
producing a gelatinous sheath surrounding the unicellular hyaline ascospores, is further 
characterized by its membranous ascomata with lanceolate setae that surround the neck, long 
cylindrical asci with a relatively large apical ring. 


KEY worDs —aquatic, Ascomycota, fungi, taxonomy 


Introduction 

Freshwater fungi are an important ecological group because of their 
indispensable function as decomposers in freshwater ecosystems (Wong et al. 
1998a); they also have potential application in producing various and useful 
bioactive secondary metabolites (Bucher et al. 2004, Zhang et al. 2008, Hosoe 
et al. 2010, Dong et al. 2011). Freshwater fungi are however, relatively poorly 
investigated comparing to their terrestrial counterpart. Until recently, freshwater 
fungi had been reported mainly from Europe, North America, and Southeast 
Asia (Cai et al. 2006b, Raja et al. 2009). Although China has a high biodiversity 
of fungi and abundant freshwater resources, investigation of freshwater fungi 
has been mostly restricted to Hong Kong and Yunnan Province. 

Annulatascaceae S.W. Wong et al. comprises many freshwater genera 
(Vijaykrishna et al. 2006). The family is characterized morphologically by dark 


82 ... Hu &al. 


brown or black ascomata with ostiolate necks, long cylindrical unitunicate 
asci with a relatively massive, refractive apical ring, and uniseriate, hyaline 
to brown, fusiform or ellipsoidal ascospores (Wong et al. 1998b, Ho & Hyde 
2000). Seventy-five species representing 21 genera are currently placed in 
Annulatascaceae (Kirk et al. 2008). 

Our research group has focused on investigating the diversity of freshwater 
fungi in China for over ten years (Hyde et al. 2000, Luo et al. 2004, Cai et al. 
2005, 2008, Hu et al. 2007, 2010a, b). During our continuing investigation, two 
undescribed ascomycetes belonging to the Annulatascaceae were collected on 
submerged wood and are described and illustrated in this paper. 


Materials & methods 

D.M. Hu, who has investigated freshwater ascomycetes on submerged woody 
material in the southern provinces of China (including Guizhou, Jiangxi and Yunnan 
provinces) since 2008, has collected 875 samples from streams, lakes, ditches, and ponds. 
After return to the laboratory, the samples were incubated following Cai et al. (2006a). 
Samples were examined for fungal fruiting bodies under a dissecting microscope 
(Leica MZ16A) during incubation. Observations and photographs were prepared from 
materials mounted in water under a compound microscope (Nikon E800). Single spore 
isolation as outlined by Choi et al. (1999) was attempted but unsuccessful. Herbarium 
specimens of the fungal taxa described in this study are deposited in the International 
Fungi Research and Development Centre (IFRDC). 


Taxonomy 


Annulatascus menglensis D.M. Hu, L. Cai & K.D. Hyde, sp. nov. PLATE 1 
MycoBank MB 563810 

Differs from other Annulatascus species by its hyaline neck. 

Type: China, Yunnan Province, Mengla, Wudaoban Stream, 21°24'N 101°36’E, alt: 660 

m, 3 April 2009, D.M. Hu (Holotype, IFRDC 023-002). 

EryMoLoecy: menglensis refers to the collecting site, Mengla, a county in southern 

China. 
AscoMaTA 180-200 high, 120-150 um in diam., subglobose to ellipsoidal, 
superficial, solitary, ostiolate, black, coriaceous. NECK 150-340 x 40-60 
um, cylindrical, hyaline, membranous, periphysate. PERIDIUM composed of 
compressed cells, outer layers dark brown, inner layers hyaline. PARAPHYSES 
ca. 5-6 um wide, broad at the base and tapering towards the apex, hyaline, 
septate, smooth-walled, simple, longer than asci. Asc1 115-163 x 10-13 um, 
8-spored, cylindrical, unitunicate, persistent, pedicellate, with a large bipartite, 
refractive, J- apical ring, ca. 3 um high, 4 um in diam. Ascosporss 21-25 x 
8-10 um (mean = 23.8 x 8.5 um, n = 30), uniseriate, overlapping, fusiform, with 
acute ends, hyaline, aseptate, guttulate, smooth, thin-walled, without sheaths or 
appendages. 


Annulatascaceae spp. nov. (China) ... 83 


PiaTE 1. Annulatascus menglensis (from holotype). a. Ascoma on submerged wood. b. Squash 
mount of an ascoma. c. Peridium comprising angular cells in surface view. d. Paraphyses. e-f. Asci. 
g. Upper part of an ascus showing the apical ring. h-k. Ascospores. Scale bars: a = 100 um, b = 50 
um, c-d = 5 um, e-f = 20 um, g-k = 5 um. 


84 ... Hu &al. 


HABITAT & DISTRIBUTION— Saprobic on submerged wood in fresh water 

in China. 

ADDITIONAL SPECIMENS EXAMINED: CHINA. YUNNAN PROVINCE: Mengla, Wudaoban 

Stream, 21°24'N 101°36’E, alt. 660 m, 3 April 2009, D.M. Hu (IFRDSC 002-017, IFRDSC 

002-018). 
COMMENTS — Annulatascus menglensis has black, coriaceous ascomata and 
cylindrical asci with a large bipartite, refractive, J- apical ring. It fits well within 
the generic concept of Annulatascus (Hyde 1992). Thus far, 14 species have been 
included in the genus (Tsui et al. 2002, Barbosa et al. 2008, Abdel-Wahab et al. 
2011). The new species resembles A. velatisporus K.D. Hyde (Hyde 1992) in 
having fusiform and aseptate ascospores. However, A. velatisporus differs from 
A. menglensis in having larger ascospores (26-42 x 9-12 um) with a gelatinous 
sheath. Annulatascus menglensis shares similar ascospore dimensions with 
A. fusiformis K.D. Hyde & S.W. Wong (Hyde & Wong 2000), A. joannae K.M. 
Tsui et al. (Tsui et al. 2002), A. citriosporus J. Frohl. & K.D. Hyde (Frohlich & 
Hyde 2000), and A. apiculatus FR. Barbosa & Gusmao (Barbosa et al. 2008) 
but lacks their black necks and mucilaginous sheaths and/or appendages. 
Annulatascus lacteus K.M. Tsui et al. (Tsui et al. 2002), which also has aseptate 
ascospores without sheath or appendage, differs from A. menglensis in having 
a milky ascomata. 


Aqualignicola vaginata D.M. Hu, L. Cai & K.D. Hyde, sp. nov. PLATE 2 
MycoBank MB 563811 

Differs from Aqualignicola hyalina in having a hyaline ascomatal neck and a gelatinous 

sheath surrounding the ascospores. 

Type: China, Yunnan Province, Mengla, Wudaoban Stream, 21°32'N E101°29’E, alt. 620 

m, 2 April 2009, D.M. Hu (Holotype, IFRDC 021-043). 

ETyMOLOoGy: vaginata (= sheath) refers to the mucilaginous sheath surrounding the 

ascospore. 
ASCOMATA 100-140 um diam, globose, membranous, brown to black-brown, 
submerged, solitary. NecK 40-60 um diam, 200-350 um long, cylindrical, 
hyaline, periphysate, covered with setae; setae 15-35 x 4-5 um, lanceolate, 
solid, surrounding the neck. PERrpr1uM 7-14 um thick, in surface view of 
textura angularis, composed of 5 layers of light-brown, pseudoparenchymatous 
cells. PARAPHYSES 4-6 tm in diam, 85-100 long, septate, flexuose, hypha-like, 
numerous, tapering towards the apex, embedded in a gelatinous matrix. AscI 
145-156 x 5.5-6.5 um, 8-spored, cylindrical, pedicellate, unitunicate, apically 
truncate, with a large, J-, refractive, apical ring. Ascospores 11-15 x 5-6 
um (mean = 13.4 x 5.3 um, n = 50), uniseriate, ellipsoidal-fusiform, hyaline, 
aseptate, thin-walled, multiguttulate, with a fan-shaped, hyaline mucilaginous 
sheath surrounding one end of the ascospores. 


Annulatascaceae spp. nov. (China) ... 85 


PLATE 2. Aqualignicola vaginata (from holotype). a. Ascoma on submerged wood. b. Section of an 
ascoma. c. Peridium. d. Section through a neck. e. Hair on neck. f. Paraphyses. g. Ascus. h. Upper 
part of an ascus, show the apical ring. i-n. Ascospores (arrows show the mucilaginous sheath and 
the multiple guttules). Scale bars: a = 100 um, b = 30 um, c = 5 um, d = 30 um, e, f= 5 um, g = 20 
um, h-n = 5 um. 


86 ... Hu & al. 


HABITAT & DISTRIBUTION — Saprobic on submerged wood in fresh water 

in China. 

ADDITIONAL SPECIMENS EXAMINED: CHINA. YUNNAN PROVINCE: Mengla, Wudaoban 

Stream, 21°32'N 101°29’E, alt. 620 m, 2 April 2009, D.M. Hu (IFRDSC 004-003, IFRDSC 

004-004, IFRDSC 004-005). 
COMMENTS — Previously monotypic, Aqualignicola V.M. Ranghoo et al. is 
characterized by membranous ascomata with a neck surrounded by lanceolate 
setae, long cylindrical asci with a relatively large apical ring, and hyaline 
unicellular ascospores (Ranghoo et al. 2001). The new species fits well with the 
generic concept of Aqualignicola but differs from the type species, A. hyalina, 
in having a hyaline ascomatal neck and a mucilaginous sheath surrounding the 
ascospore. Furthermore, the ascospores and asci of A. vaginata are narrower 
than those of A. hyalina (ascospores: 14-15 x 6.25-7.5 um; asci 9-12 um diam.). 
Aqualignicola vaginata is also similar to species of Aquaticola W.H. Ho et al. 
(Ho et al. 1999) and Longicollum Zelski et al. (Zelski et al. 2011a). However, the 
species of the latter two genera have glabrous ascomata and lack the lanceolate 
setae surrounding the neck found in A. vaginata. Aqualignicola vaginata can 
be compared to the newly described Chaetorostrum quincemilense Zelski et al. 
(Zelski et al. 2011b) in that both taxa have a hyaline neck covered with setae; 
however the ascospores of A. vaginata are aseptate and much smaller than 
those of C. quincemilense (1-3-septate; 30-38 x 10-12 um). 


Acknowledgments 

Funds for research were provided by a major project of the National Natural Science 
Foundation of China, No. 31093440. The Global Research Network for Fungal Biology 
and King Saud University are thanked for support. 


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Ranghoo VM, Tsui CKM, Hyde KD. 2001. Brunneosporella aquatica gen. et sp. nov., Aqualignicola 
hyalina gen. et sp. nov., Jobellisia viridifusca sp. nov. and Porosphaerellopsis bipolaris sp. nov. 
(ascomycetes) from submerged wood in freshwater habitats. Mycological Research 105: 
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Tsui CKM, Ranghoo VM, Hodgkiss IJ, Hyde KD. 2002. Three new species of Annulatascus 
(Ascomycetes) from Hong Kong freshwater habitats. Mycoscience 43: 383-389. 
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Vijaykrishna D, Jeewon R, Hyde KD. 2006. Molecular taxonomy, origins and evolution of freshwater 
ascomycetes. Fungal Diversity 23: 351-390. 

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Yuen TK. 1998a. Role of fungi in freshwater ecosystems. Biodiversity and Conservation 7: 
1187-1206. http://dx.doi.org/10.1023/A:10088837 16975 


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Zhang KQ, Wang L, Dong JY, Song HC, Shen KZ, Wang LM, Sun R, Wang CR, Li GH, Li L. 2008. 
Screening and isolation of antibacterial activities of the fermentative extracts of freshwater 
fungi from Yunnan Province, China. Annals of Microbiology 58: 579-584. 
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Zelski SE, Raja HA, Miller AN, Barbosa FR, Gusmao LFP, Shearer CA. 2011la. Longicollum 

biappendiculatum gen. et sp. nov., a new freshwater ascomycete from the Neotropics. 
Mycosphere 2: 539-545. 
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a new freshwater ascomycete and its Taeniolella-like anamorph from Peru. Mycosphere 2: 
593-600. 


ISSN (print) 0093-4666 © 2012. Mycotaxon, Ltd. ISSN (online) 2154-8889 


MY COTAXON 


http://dx.doi.org/10.5248/120.89 
Volume 120, pp. 89-98 April-June 2012 


New records of little-known species of Carbomyces 
(Carbomycetaceae, Ascomycota) 


GABRIEL MORENO”™*, MARCOS LIZARRAGA’*, MARTIN ESQUEDA3, 
RICARDO GALAN’ & PABLO ALVARADO’ 


' Dpto. de Biologia Vegetal, Facultad de Biologia, Universidad de Alcala, 
Alcala de Henares, Madrid 28871 Spain 
? Dpto. de Ciencias Quimico-Biologicas, Programa de Biologia, Universidad Auténoma de Ciudad 
Juarez, Anillo Envolvente Pronaf y Estocolmo s/n, 
Ciudad Juarez, Chihuahua 32300 México 
* Centro de Investigacion en Alimentacion y Desarrollo, A.C., 
Apartado Postal 1735, Hermosillo, Sonora, C.P. 83000 México 
* CORRESPONDENCE TO: gabriel.moreno@uah.es 


ABSTRACT— ‘The genus Carbomyces is reported for the first time for the Mexican mycobiota. 
Carbomyces emergens, C. gilbertsonii, and C. longii were collected in the Chihuahuan desert, 
Mexico. Carbomyces emergens was more frequently collected and more widely distributed, 
while C. gilbertsonii and C. longii each have a restricted distribution and represent second 
records worldwide. The phenology, chorology, and macro-/microscopic characters (including 
spore ornamentation by SEM) are outlined for all species. 


Key worps— Pezizales, taxonomy, truffles 


Introduction 

Several collections of hypogeous and semi-hypogeous species have been 
recorded in recent studies of gasteroid and secotioid fungi in Chihuahua State, 
including the central Chihuahuan desert, Mexico (Lizarraga et al. 2010; Moreno 
et al. 2010). Fungi reported here were found on the soil surface and wrongly 
considered in situ to represent basidiomycetes. However, careful examination 
revealed that these fungi represent two different ascomycete species in the 
genus Carbomyces Gilkey. 

Hypogeous ascomycetes, which are widely distributed, occur in different 
forest types (e.g., coniferous dominated by Pinaceae, angiospermous with 
Betulaceae, Salicaceae, Fagaceae, and Myrtaceae). Some even associate with 
herbaceous plants and shrubs, such as those in the Cistaceae. Cistaceous plants 
found in arid areas of the Mediterranean basin, southern Europe, northern 


90 ... Moreno & al. 


Africa, and the Middle East play an important role in plant conservation by 
protecting sensitive areas from erosion from heavy rain and human activity. In 
these areas, cistaceous plants can form mycorrhizal associations with several 
different fungal genera such as Choiromyces, Picoa, Terfezia, Tirmania, and 
Tuber. 

However, in America, Cistaceae does not occur and arid lands are commonly 
dominated by Cactaceae, Fouquieriaceae, Mimosaceae, and Zygophyllaceae. 
Carbomyces was described from deserts of New Mexico and California (USA) 
for C. emergens and C. longii by Gilkey (1954), who published macro- and 
microscopical drawings of both species (Gilkey 1955). 

Trappe & Weber (2001) later described in detail those two species and 
transferred a third species, C. gilbertsonii, into Carbomyces from the puffball 
basidiomycete genus Abstoma G. Cunn. where it had previously been misplaced 
(Zeller 1944). These truffles appear endemic to North American deserts, 
occurring in the Chihuahuan, Sonoran, and Mohave deserts from New Mexico 
to southern California, where they appear from September through April. 
Carbomyces, which is adapted to extreme xeric conditions, differs from other 
hypogeous ascomycetes in globose emergent ascomata, a two-layered peridium, 
and a powdery gleba at maturity resulting from the disintegrated asci (a feature 
also shared with Elaphomyces spp. and Ruhlandiella berolinensis Henn.). 

Most specimens have been found ex situ since they are easily wind-blown 
when mature (lying dry and loose) or eaten and transported (when immature?) 
by rodent mycophagists (Zak & Whitford 1986), so that fungal populations 
can be easily intermixed and subsequently confused. Nevertheless, in the 
Chihuahuan desert (Mexico), we found pure (i.e. non-intermixed) populations 
in situ that appeared macroscopically very similar, to other known species based 
on spore ornamentation (smooth vs. rough) and shape (ellipsoid vs. globose). 

Gilkey (1954) placed Carbomyces in the Terfeziaceae, but Trappe (1971) 
erected the monotypic Carbomycetaceae for the genus, a taxonomic position 
later accepted by several authors (Trappe 1979, Trappe & Weber 2001, Lumbsch 
& Huhndorf 2007). Nevertheless, the true relationship between Carbomyces and 
other ascomycetes was unknown until recently (Hansen & Pfister 2006). The 
putatively non-mycorrhizal genus Carbomyces is thought to be ectomycorrhizal 
(ECM), although no ECM hosts have been identified in its natural habitat 
(Trappe 1971; Zak & Whitford 1986; Trappe & Weber 2001). 

In the present workall three Carbomyces species —C. emergens, C. gilbertsonii, 
C. longii— are reported from Mexico for the first time and redescribed from 
recently collected specimens. 


Materials & methods 
The ascocarps were studied in situ following general methods (Cifuentes et al. 
1986, Moreno & Manjén 2010). Dried material was studied at the lab using KOH 5%, 


Carbomyces in Mexico ... 91 


Melzer’s reagent (MLZ), Cotton blue (CB), Congo red 1%, and Hoyer’s medium for 
microscopical observations. A Nikon Eclipse 80i phase-contrast microscope was used 
for spore measurements (on dead dried material) under oil immersion including surface 
structures such as spines or warts (on 25 spores). 

Spore ornamentation was also studied under a scanning electron microscope Zeiss 
DSM 950. Critical point drying technique was performed on the samples prior to 
mounting, following Moreno et al. (1995). 

The collections are kept in the Herbarium UAC] of the “Departamento de Ciencias 
Basicas, Universidad Autonoma de Ciudad Juarez,’ Mexico, with duplicate sets in 
Herbarium AH of the Universidad de Alcala de Henares and the macromycetes 
collection of the Centro de Estudios Superiores del Estado de Sonora (CESUES). 

High-quality DNA extract could be obtained from only one fruiting body of 
Carbomyces emergens (AH 39344), probably due to the extreme climatic conditions and 
high temperatures over long periods to which the other samples were naturally exposed. 
DNA extraction and PCR protocols followed Checa et al. (2012). ITS (JQ023163) and 
28S nLSU (JQ023164) regions were sequenced, deposited in GenBank, and aligned 
with their closest BLAST matches from sequences deposited by Ferdman et al. (2005), 
Hansen et al. (2001, 2005), Trappe et al. (2010), and others. 


Taxonomy 


Carbomyces emergens Gilkey, N. Amer. Fl., Ser. 2, 1: 27 (1954) FIGS 1-3, 11-13 
Ascomata subglobose, ovoid to pyriform, 1.2-3 x 1.5-4.4 cm, scattered or 
occasionally in small groups. PERIDIUM coriaceous, two-layered: outer layer 
very thin, membranous, initially whitish but finally yellow-brownish to dirty 
greyish, firmly attached, cracking by fissures or polygonal plates; inner layer 
ochraceous to dark brown, 0.2-0.6 mm thick. GLEBA multilocular, although 
apparently seeming to be solid, dark brown with sterile, lighter veins forming 
a fragile reticule; spore mass pulverulent, dark with olivaceous or vinaceous 
tints. Asci globose to subglobose or ovoid, 8-spored, 40-55 x 30-5 um, with 
thin brownish walls disintegrating at maturity. Ascospores hyaline to light 
yellowish, globose, inamyloid, 9-14 um diam., having a thick wall (1.2-1.5 um) 
finely punctate when fully mature (as seen at 400x under LM, feebly verrucose 
under the SEM), but appearing smooth when immature. 
SPECIMENS EXAMINED: MEXICO, Cuinvuanua: Municipality of Juarez, in arid land: 
20.IV.2005 R. Silva (UACJ 06); km 30 road Ciudad Juarez to Casas Grandes, next to 
Larrea tridentata Coville, 3.VIII.2008 M. Lizarraga, H. Pelayo (UAC] 1166 in AH 39344; 
GenBank Acc. Nos.—ITS: JQ023163, LSU: JQ023164); 31°23’20.21”N 106°23°32.94” W, 
alt. 1312 m, 27.[I1.2010, M. Lizarraga, M. Vargas, D. Lopez (UACJ 1555); 8.V.2010 
M. Lizarraga, D. Lopez, F. Félix, M. Vargas (UACJ 1556); 15.1X.2010 (UACJ 1684); 
6.X1.2010 M. Vargas, M. Lizarraga (UACJ 1687); 31°22’47.44”°N 106°24°3.89"W, alt. 
1319 m, 22.X.2010 (UACJ 1688; 30.X.2010 (UACJ 1700); 6.XI.2010 (UACJ 1689); 
31°20°13.47°N 106°23°32.95"W, alt. 1334 m, 15.V.2010 M. Vargas, F. Félix, M. Lizarraga, 
D. Lopez (UACJ 1686); 15.1X.2010 M. Vargas, M. Lizarraga (UAC] 1685); 10.IV.2011 
(UACJ 1683); 31°19°39.98”N 106°30°7.43”W, alt. 1280 m, 23.V.2010 M. Lizarraga, D. 


92 ... Moreno & al. 


Lépez, M. Vargas (UAC] 1557); 2.X.2010 M. Lizarraga, M. Vargas, D. Saenz (UAC] 

1628). Municipality of Ojinaga, La Mula, 29°14’24.0”N 104°26°09.0"W, alt. 1380 m, 

24.V.2009 M. Lizarraga, C. Salazar, D. Saenz, D. Lopez, A. Gutierrez, E. Hernandez (HM 

22 in CESUES 7550); 24.X.2009 M. Lizarraga, A. Sanchez, A. Gutierrez, C. Salazar, D. 

Lopez (HM 171 in CESUES 7551). 
REMARKS— Carbomyces emergens has been repeatedly described (e.g., Trappe 
& Weber 2001) as having smooth or minutely roughened ascospores, but our 
studies show they are clearly warted at or above 400x. This may be because 
fully mature ascomata had not been found until now. The species could be 
macroscopically confused with C. longii and C. gilbertsonii, which share a 
similar habitat, but the species are easily differentiated by shape and spore 
ornamentation. Small sporocarps of C. emergens could easily be misidentified 
in situ as species of the basidiomycete genus Arachnion Schwein. (Agaricaceae) 
but in Arachnion the gleba lacks ascal remnants and contains a capillitium, and 
small (< 6 um diam.) verrucose spores. 

Zak & Whitford (1986) described the hypogeous development of Carbomyces 
emergens and reported the ascomata’s apparent ingestion by rodents after 
collecting a few dried sporocarps in Northern Chihuahuan desert. Despite 
firm evidence, they suggested that fruiting probably occurs during late winter 
(January-March). Our collections UACJ 06 and UACJ 1557 are assumed 
to be immature, while the remaining specimens show an evident mature 
development. 

Trappe & Weber (2001) regard C. emergens as the most common and 
widely distributed Carbomyces species, although reported only from American 
Southwest —Arizona, California, and New Mexico. Here we report it for the 
first time in Mexico as an apparently common species of the central Chihuahuan 
desert. 


Carbomyces gilbertsonii N.S. Weber & Trappe, Harvard Pap. Bot. 6: 212 (2001) 
[nom. nov.] FIGs 4-7, 14-16 
= Abstoma longii Zeller, Mycologia 36: 628 (1944) [non Carbomyces longii Gilkey] 

ASCOMATA immersed and finally superficial, globose to subglobose, 
laterally compressed to irregular, 2-3 cm diam. PERIDIUM two-layered, mostly 
persistent, ferruginous at maturity. GLEBA solid, pulverulent, brown to dark 
brown. Ascr subglobose, 8-spored, 40-65 um diam., with thin light brown walls 
disintegrating at maturity. Ascosporgs hyaline to brownish with age, globose 
to subglobose, inamyloid, 20-25 um, ornamented with spine-like elements up 
to 2 um long (LM). Under SEM such “spines” consist in small clumps of slender 
apically fused baculae, forming pyramids with hollow bases. 

SPECIMENS EXAMINED: MEXICO, CurHuaunvua: Municipality of Juarez, 31°22’47.44"N 


106°24°3.89" W, alt. 1319 m, in arid soil near to Larrea tridentata, Prosopis sp. and 
Opuntia sp., 23.X.2010 M. Lizarraga, M. Vargas, (UACJ 1559, duplicate in AH 39346); 


Carbomyces in Mexico ... 93 


Fics. 1-3: Carbomyces emergens AH 39344: 1. Ascocarps. 2. Asci with spores. 3. Spores (SEM). 
Fics. 4-7 Carbomyces gilbertsonii AH 39347: 4. Ascocarps. 5. Asci with spores. 6. Free spores. 7. 
Spores (SEM). Fics. 8-10 Carbomyces longii AH 39345: 8. Ascocarps. 9. Free spores. 10. Spore 
(SEM). Scale bars: 1, 4, 8 = 1 cm; 2, 5, 6, 9 = 10 um; 3, 10 = 5 um; 7 = 10 um. 


6.XI.2010, (UACJ 1561 in AH 39347); (UACJ 1691); 31°23°20.21”N 106°23’32.94°W, alt. 

1312 m, (UACJ 1690); 31°22’21.46”N 106°33’59.26”W, alt. 1225 m, 26.III.2011 (UACJ 

1689). 
REMARKS— Carbomyces gilbertsonii could be macro- and microscopically 
confused with C. longii, but small, ellipsoid spores (17-19 x 12-13 um) 
distinguish the latter species. Old Scleroderma cepa specimens could be 
confused with C. gilbertsonii specimens without ascal remains, but the smaller 
(9-12 um) spores of S. cepa spores are diagnostic. It is noteworthy that 


94 ... Moreno & al. 


Fics. 11-13: Carbomyces emergens AH 39344: spores (SEM. Fics. 14-16 Carbomyces gilbertsonii 
AH 39347: 14-15. Spores (SEM); 16. Spore ornamentation (detail, SEM). Fics. 17-19 Carbomyces 
longii AH 39345: 17-18. Spores under SEM. 19. Spore ornamentation (detail, SEM). Scale bar: 
11-13, 16 = 2 um; 14-15, 17-18 = 5 um; 19 = 1 um. 


C. gilbertsonii was previously known only from the type locality (New Mexico, 
U.S.A.); our collections add Chihuahua (Mexico) to its distribution. 


Carbomyces longii Gilkey, N. Amer. Fl., Ser. 2, 1:27 (1954) FIGs 8-10, 17-19 

Ascomarta subglobose, laterally compressed, pyriform to irregular, 2-3 cm 
diam., 2.5-3 x 1.5-2 cm. PERIDIUM two-layered, persistent, outer layer white to 
greyish, later light brown to dark brown with reddish tinges at maturity. GLEBA 
pulverulent, dark brown to brown, becoming olivaceous. Asci evanescent, only 
a few fragments of brownish, disintegrated walls were seen. ASCosPoREs light 
yellowish, ellipsoid, 17-19 x 12-13 um, inamyloid, ornamented with regularly 


Carbomyces in Mexico ... 95 


distributed spines < 1 um long (LM). Under SEM, the spines are scattered, 
usually with bent apices. 


SPECIMENSEXAMINED: MEXICO, CHIHUAHUA: Municipality of Ahumada, 31°07'32.0”N 

106°29°48.0"W, alt. 1280 m, in soil next to Larrea tridentata, 24.V1I.2009 M. Lizarraga, 

C. Salazar, D. Saenz, D. Lopez, A. Gutierrez, E. Hernandez (UAC] 1699). Municipality of 

Juarez, 31°23°20.21”N 106°23°32.94"W, alt. 1312 m, 27.III.2010 M. Lizarraga, M. Vargas, 

D. Lopez (UACJ 1557 in AH 39017); 3.V.2010 (UACJ 1698); 15.V.2010 M. Lizarraga, 

D. Lopez, M. Vargas, EF. Félix (UACJ 1558); 15.1X.2010 M. Lizarraga, M. Vargas (UAC] 

1697); 22.X.2010 (UAC] 1696); 6.XI.2010 (UAC] 1694); 31°22’47.44”N 106°24°3.89" W, 

alt. 1319 m, 30.X.2010 (UACJ 1562, duplicate in AH 39345); 6.X1.2010 (UACJ 1693); 

31°22’21.46"N 106°33’59.26”W, alt. 1225 m, 26.11.2011 (UACJ 1695). 
REMARKS— Since C. longii asci are fully evanescent, mature sporocarps could 
be confused with Arachniopsis albicans Long (Agaricaceae), but the latter is 
distinguished by small (3-5 x 3-4 um) oval feebly verrucose basidiospores and 
a hyaline capillitium. Co-occurring with C. gilbertsonii, C. longii was previously 
known only from the type locality (New Mexico, U.S.A.). Our report from 
Chihuahua, Mexico, represents the second world record. 


Key to Carbomyces species in the Chihuahuan desert, Mexico 
1 Spores globose and smooth to subsmooth (LM) or feebly warted (SEM) ....C. emergens 
NPR PoC anG PETY Fe crac lh ees AM chai ah Aaa a chai OM eh e ohdr at eed a dee gM lec, eats eee 2 


2-Spores-ellipsoidsscatiered:Spities, 5 2 fern, Horne, hess le lene ele ng geen a C. longii 


2’ Spores globose to subglobose, spines in small clumps forming pyramids 
ee ee tee PRR PMs Rieko Rumen PRLS PR Uh Wire ota C. gilbertsonii 


Molecular results 

28S nLSU alignment comprised 39 sequences with 215 variable sites among 
563 bases, 133 of them parsimony-informative. Bayesian and parsimony 
analyses were generally consistent with each other (Fig. 20) and supported the 
three main groups already recognized in Lzessoe & Hansen (2007). The first 
group comprises Kalaharituber, Iodowynnea, Hydnotryopsis, Sarcosphaera, 
and several Peziza spp. The second group includes Mattirolomyces, Elderia, 
and a different set of Peziza spp. The third group includes Terfezia, Tirmania, 
Mycoclelandia, Ulurua, Eremiomyces, Ruhlandiella, Hapsidomyces, Plicaria, and 
a third set of Peziza spp. The analyses show Carbomyces as somewhat related 
to Kalaharituber (Pezizaceae) in the first group (Fig. 20), partially confirmed 
by BLAST of the only ITS sequence obtained, which linked C. emergens to 
Terfezia and Kalaharituber (84% and 83% identity in 68% and 65% coverage, 
respectively), in accordance with data obtained by Karen Hansen (pers. 
comm.). 


96 ... Moreno & al. 


AY544677 Ascobolus carbonarius 
AF335148 Peziza luteoloflavida 
AF335159 Peziza retrocurvata 
AF335156 Peziza phyllogena 
AY789328 Peziza phyllogena 
gg 7 AY500533 Hapsidomyces venezuelensis 
100 L_ AY500553 Plicaria anthracina 
400 JN032129 Eremiomyces magnisporus 
100 AF435823 Eremiomyces echinulatus 
AF335175 Ruhlandiella berolinensis 
“a GQ231750 Ulurua nonparaphysata 
100 GQ231747 Mycoclelandia bulundari 
79 GQ231743 Mycoclelandia arenacea 
109 es AF335135 Peziza depressa 
100 AF335132 Peziza badia 


PL AF335147 Peziza limnaea 
95 AF 499448 Terfezia boudieri 
100 AF435824 Terfezia claveryi 
94 AF335177 Tirmania nivea 


100 L__ AF335178 Tirmania pinoyi 
AY500548 Peziza lobulata 
AF335165 Peziza subviolacea 
GQ231753 Mattirolomyces austroafricanus 
ae HQ660382 Mattirolomyces spinosus 
400 7 GQ231734 Elderia arenivaga 
100 L GQ231737 Elderia arenivaga 


71 4100 AF335146 Peziza howsei 


Po 100 L AF335140 Peziza emileia 
99 AF335158 Peziza proteana 
100 AY500545 Peziza exogelatinosa 
ats AY500552 Peziza vesiculosa 
100 a AF335154 Peziza nivalis 


100 AF335145 Peziza fimeti 
AF335174 Pfistera pyrophila 
UACJ 1166 Carbomyces emergens 
AY232726 Kalaharituber pfeilii 

AF335118 lodowynnea auriformis 

400 AF335115 Hydnotryopsis setchellii 

100 NG 027607 Sarcosphaera crassa 

ob AY500550 Peziza obtuspiculata 


100 | AF335157 Peziza polaripapulata 0.05 


Carbomyces in Mexico ... 97 


Acknowledgements 

The authors thank CONABIO (GTO016 project) for the financial support of this 
study. One of the authors (M. Lizarraga) extends his gratitude to the collectors and the 
“Vicerrectorado de Investigacién e Innovacién” of the Universidad de Alcala for their 
assistance in obtaining financial support for his research stage during two months at 
the Universidad de Alcala. We express our gratitude to Dr. G. Pacioni and Lda. M?.M. 
Dios for reviewing the manuscript and adding a number of useful comments. We wish 
to express our gratitude to Mr. D.W. Mitchell for his assistance in correcting the English 
and to Mr. A. Priego and Mr. J.A. Pérez (Electron Microscopy Service, University of 
Alcala) for their invaluable help with the SEM. We also thank L. Monje and A. Pueblas 
of the “Gabinete de Dibujo y Fotografia Cientifica” at the Universidad de Alcala for their 
invaluable help in the digital preparation of the photographs. We are also grateful to Dr. 
J. Rejos, curator of the AH herbarium for providing specimens management. Finally we 
would like to thank Dr. Karen Hansen for her useful comments on the results and Aldo 
Gutiérrez (CIAD) for formatting the text. 


Literature cited 

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a new species and its anamorph state described and sequenced from Europe. Mycological 
Progress. http://dx.doi.org/10.1007/s11557-011-0776-8 

Cifuentes J, Villegas M, Pérez-Ramirez L, Sierra S. 1986. Hongos. 55-64, in: A Lot, F Chiang (eds). 
Manual de herbario. Consejo Nacional de la Flora de México, A.C., México. 

Ferdman Y, Aviram S, Roth-Bejerano N, Trappe JM. 2005. Phylogenetic studies of Terfezia pfeilii 
and Choiromyces echinulatus (Pezizales) support new genera for southern African truffles: 
Kalaharituber and Eremiomyces. Mycological Research 109: 237-245. 
http://dx.doi.org/10.1017/S0953756204001789 

Gilkey HM. 1954. Tuberales. North American Flora 2, 1: 1-36. 

Gilkey HM. 1955 [“1954”]. Taxonomic notes on Tuberales. Mycologia 46: 783-793. 

Hansen K, Pfister DH. 2006. Systematics of the Pezizomycetes - the Operculate Discomycetes. 
Mycologia 98: 1029-1040. http://dx.doi.org/10.3852/mycologia.98.6.1029 

Hansen K, Laessge T, Pfister DH. 2001. Phylogenetics of the Pezizaceae, with an emphasis on 
Peziza, Mycologia 93: 958-990. http://dx.doi.org/10.2307/3761760 

Hansen K, LoBuglio KF, Pfister DH. 2005. Evolutionary relationships of the cup-fungus genus 
Peziza and Pezizaceae inferred from multiple nuclear genes: RPB2, B-tubulin, and LSU rDNA. 
Molecular Phylogenetics and Evolution 36(1): 1-23. 
http://dx.doi.org/10.1016/j.ympev.2005.03.010 

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Agaricaceae) en la Planicie Central del Desierto Chihuahuense. Rev. Mex. Micol. 32: 17-23. 


Fic. 20: 28S nLSU consensus phylogram constructed in MrBayes 3.1 showing the most probable 
relationships between Carbomyces emergens and its closest relatives in the Pezizaceae. Numbers close 
to nodes represent maximum parsimony bootstrap proportions (upper), and Bayesian posterior 
probabilities (lower). Only nodes supported by at least one inference method were annotated. 


98 ... Moreno & al. 


Lumbsch HT, Huhndorf SM. 2007. Outline of Ascomycota - 2007. Myconet 13: 1-58. 

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Zak JC, Whitford WG. 1986. The occurrence of a hypogeous ascomycete in the northern 
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http://dx.doi.org/10.2307/3754840 


ISSN (print) 0093-4666 © 2012. Mycotaxon, Ltd. ISSN (online) 2154-8889 


MY COTAXON 


http://dx.doi.org/10.5248/120.99 
Volume 120, pp. 99-103 April-June 2012 


Cantharellus zangii, a new subalpine basidiomycete 
from southwestern China 


XIAO-FEI TIAN}, BART BuyCK‘, SHI-CHENG SHAO””, 
Pei-Guti Liu’ & YAN FANG> 


' Key Laboratory of Biodiversity and Biogeography, Kunming Institute of Botany, 
Chinese Academy of Sciences, 132 Lanhei Road, Kunming,650204, China 
? Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, 
88 Xuefu Road, Kunming, 650223, China 
> Graduate University of Chinese Academy of Sciences, 
19(A) Yuquan Road, Beijing, 100049, China 
* Muséum national d’histoire naturelle, Département de Systématique et Evolution, 
Case Postale n°39. UMR 7205, 57, Rue de Cuvier, Paris, F- 75231, France 
° Department of Analysis, Zibo Water Supply, 14 Gonggingtuan East, Zibo, 255000, China 
*CORRESPONDENCE TO: pgliu@mail.kib.ac.cn 


ABSTRACT — Cantharellus zangii is described and illustrated from subalpine forest (>3000 m) 
in northwestern Yunnan, China. The diagnostic characteristics of C. zangii are the small thin- 
fleshed fruit bodies with a long tapering fistulose stipe, a bright orange veined hymenophore, 
thin-walled clamped hyphae, and large ellipsoid basidiospores. So far it is only known from 
the type locality and its nearby area. 


KEY worpDs — taxonomy, endemic species, ectomycorrhizal, Cantharellales 


Introduction 

Cantharellus Adans. ex Fr., the type genus of Cantharellaceae, was 
established by Fries (1821) and later typified with C. cibarius Fr. by Earle (1905). 
Thirteen species of Cantharellus have previously been reported from China: 
C. appalachiensis R.H. Petersen, C. buccinalis Mont., C. cibarius, C. cinereus 
(Pers.) Fr., C. cinnabarinus (Schwein.) Schwein., C. friesii Quél., C. lateritius 
(Berk.) Singer, C. minor Peck, C. patouillardii Sacc., C. subalbidus A.H. Sm. 
& Morse, C. tuberculosporus M. Zang, C. vaginatus S.C. Shao et al., and 
C. yunnanensis W.F. Chiu (Teng 1963, Chiou 1973, Zang 1980, Zhuang 2001, 
2005, Tian et al. 2009, Shao et al. 2011). However, some of these collections are 
probably misidentified and need to be verified microscopically and molecularly, 


100 ... Tian & al. 


as has been done by Buyck et al. (2011) and Buyck & Hofstetter (2011) for 
North America. During our Cantharellus research, we came across one small 
species with a very slender stipe in the subalpine mixed forest of Shangri-La 
(northwestern Yunnan, China), which we determined as distinct from already 
described species in the genus. It is here described as Cantharellus zangii. 


Materials & methods 

Macro-morphological fruitbody features were recorded and photographed in the 
field; color designations and codes follow Kornerup & Wanscher (1961). In basidiospore 
measurements, [n/m/p] = n basidiospores measured from m basidiomata of p collections 
in 5% KOH solution, with dimensions given as (a—)b-c(—d), where a and d are extremes 
and b-c represent the interval range of 90% measured values. Q = basidiospore length/ 
width ratio; Q. = average Q of all basidiospores + standard deviation. Herbarium 
abbreviation HKAS = Herbarium of Cryptogams, Kunming Institute of Botany, Chinese 
Academy of Sciences. 


Taxonomy 


Cantharellus zangii X.F. Tian, P.G. Liu & Buyck, sp. nov. PLATE 1-5 
MycoBank MB 563725 
Differs from Craterellus tubaeformis by a plano-convex pileus and bright orange 
hymenium and from Cantharellus queletii in the fistulose stipe and hymenium with 
branching veins. 


Type: China, northwestern Yunnan, Shangri La, Bitahai National Natural Reserve, 
9.IX.2008, X.F Tian 417 (Holotype, HKAS55791). 


ETYMOLOGY: zangii is in honor of Professor Zang Mu (28 Dec. 1930-10 Nov. 2011), 

a distinguished Chinese mycologist who founded the Cryptogamic Herbarium in 

Kunming Institute of Botany, Academia Sinica (KUN-HKAS), and pioneered the study 

of cryptogam diversity in southwestern China. 
MACROCHARACTERS — _ BASIDIOMATA small to medium, _ slender, 
submembranaceous, campanuliform to infundibuliform. PrLeus 2-3 cm in 
diam, at first umbonate then plane or slightly concave; squamules in the middle; 
ochre to ochre yellow (4B8-4B6), with tiny cinereous to grey (4B8-4B6); 
margin hygrophanous, smooth, with streaks, sometimes undulate, incurved at 
first, then straight. Stipe 7-8 x 0.3-0.4 cm, cylindrical to laterally compressed, 
sometimes slightly curved, attenuate towards base; entirely fistulose; dark 
orange-yellow (4B5-4B6) to bright orange (4A6). LAMELLAE decurrent, 
golden; 1.5-2 mm high, 1-2 mm apart, with transverse venations, with short 
branched lamellulae near the edge, seldom anastomosing. CONTEXT about 0.2 
cm thick in the cap center; dull white (1A2), fibrous. ODoR Osmanthus-like. 
TASTE mild. 

MICROCHARACTERS — BASIDIOSPORES [85/4/2] (8—)8.5-11 x (4.5-)5-6.5 

(-7) um, Q = (1.3-)1.4-2(-2.2), Q. = 1.7 + 0.2, ellipsoid or adaxially slightly 


Cantharellus zangii sp. nov. (China) ... 101 


FiGurRE 1-5. Cantharellus zangii (HKAS 55791, holotype): 1. Fruit bodies. 2. Hyphal extremities at 
the cap surface. 3. Tramal hyphae with clamps. 4. Basidiospores, often with many tiny oil drops. 5. 
Hymenium (with lengthening basidioles and basidia). 


102 ... Tian & al. 


depressed and more or less reniform; thin-walled, hyaline, nearly colorless, 
sometimes with tiny oil drops. Basrp1a 75-85 x 6-9 um, slender, clavate, 
5(-6)-spored; sterigmata 5-6 um long. CysTip1A absent. LAMELLAR TRAMA 
irregular, composed of colorless and loose hyphae, 3-5 um diam. PILEIPELLIS 
a layer composed of mostly radially arranged hyphae, slightly brownish, 7-8 
um in diam; the terminal cell more or less clavate; CLAmps very distinct and 
abundant in all parts. 

ECOLOGY & DISTRIBUTION — Single or in groups on the ground in subalpine 
(>3000m) mixed forests dominated by Larix potaninii var. macrocarpa 
Y.W. Law and Picea likiangensis (Franch.) E. Pritz. So far known only from 
Shangri-La subalpine area, northwestern Yunnan, China. 


ADDITIONAL SPECIMENS EXAMINED — CHINA. NORTHWESTERN YUNNAN, Shangri- 
La: Big Ravine, alt. 3030 m, 16.IX.2007, Y.C. Li 1537 (HKAS 55743); Haba Snowy 
Mountains, alt. 3000 m, 30.1X.2007, Feng Bang 182 (HKAS 55824). 


Discussion 

Based on its almost membranous pileus and veins as well as the hollow 
stipe, Cantharellus zangii could be taken for Craterellus tubaeformis (Fr.) Quel. 
(Redhead et al. 2002) at first glimpse. However, C. zangii often has an umbrella- 
shaped slender fruit body with a centrally plane or protruding pileus, whereas 
Cr. tubaeformis is horn-shaped with centrally depressed caps and has a grey or 
grey-white hymenium, never bright orange like C. zangii. 

The membranous cap and slender stipe of Cantharellus zangii are also similar 
to the European C. queletii (Ferry) Corner (Corner 1966), which differs in a 
somewhat solid stipe and the absence of branching veins in the hymenium. 

Cantharellus zangii also has some striking similarities with a few of the 
smaller American species (Petersen & Ryvarden 1971; Feibelman et al. 1996; 
Buyck et al. 2010). The yellow C. minor, for instance, shares the exceptional 
character of the fistulose stipe and thin-walled, voluminous hyphae in the 
pileipellis. However, its overall size is smaller, it also has slightly smaller spores, 
and the terminal cells in the pileipellis usually narrow at the extreme tips. The 
yellowish brown C. tabernensis Feib. & Cibula is similar in size to C. zangii 
with a very similar pileipellis, but the stipe is not hollow and the spores are 
much smaller. The same is true for C. appalachiensis, a more brownish and 
slightly more robust sister-species of C. tabernensis, possessing an identical 
microscopy. 

The small size and abundant clamps together with the thin-walled hyphal 
extremities in the cap place this species in C. subgenus Parvocantharellus as 
defined by Eyssartier & Buyck (2001), where it is further diagnosed by its large 
spores and clavate terminal cells in the pileipellis. 


Cantharellus zangii sp. nov. (China) ... 103 


Acknowledgments 

We wish to express our gratitude to Professor R. Petersen (University of Tennessee) 
and Professor M. Verbeken (Ghent University) for reviewing the manuscript. Thanks 
also given to Prof. Z.Y. Su and Dr. Z.L. Yang (Kunming Institute of Botany, Chinese 
Academy of Sciences) and Dr. S. Pennycook (Manaaki Whenua Landcare Research, 
New Zealand) for valuable suggestions. This work was supported by the National 
Natural Science Foundation of China (No. 30770007 and 30800005), the Joint Funds 
from Chinese National Sciences Foundation and Yunnan Province Government (No. 
U0836604), Natural Science Foundation of Yunnan (Key project No.2007C0002Z) and 
Foundation of Key Laboratory of Biodiversity and Biogeography, Kunming Institute of 
Botany, CAS (No.2008004), as well as Yunnan International Collaborative Program of 
innovation to strong provinces by Science and Technology (2009AC013). 


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Cantharellus cibarius complex in the southeastern USA. Fungal Diversity 49: 35-46. 
http://dx.doi.org/10.1007/s13225-011-0095-z 

Buyck B, Lewis DP, Eyssartier G, Hofstetter V. 2010. Cantharellus quercophilus sp. nov. and its 
comparison to other small, yellow or brown American chanterelles. Cryptogamie, Mycologie 
bl 17—33: 

Buyck B, Cruaud C, Couloux A, Hofstetter V. 2011. Cantharellus texensis sp. nov. from Texas, 
a southern lookalike of C. cinnabarinus revealed by tef-1 sequence data. Mycologia 103: 
1037-1046. http://dx.doi.org/10.3852/10-261 

Chiou WE. 1973. Ten new species of Agaricales from Yunnan, China. Acta Microbiologica Sinica 
13(2): 129-135. 

Corner EJH. 1966. A monograph of cantharelloid fungi. Oxford University Press. London. 255 p. 

Earle FS. 1905. The genera of the North American gill fungi. Bulletin of the New York Botanical 
Garden 5: 373-383. 

Eyssartier G., Buyck B. 2001. Novitates. Note nomenclaturale et systématique sur le genre 
Cantharellus. Documents mycologiques 31(121): 55-56. 

Feibelman TP, Bennett JW, Cibula WG. 1996. Cantharellus tabernensis: a new species from the 
southeastern United States. Mycologia 88: 295-301. http://dx.doi.org/10.2307/3760934 

Fries EM. 1821. Systema Mycologicum. Vol.1. Lundae. 

Kornerup A, Wanscher JH. 1961. Farver i Farver. Politikens Forlag. Kobenhavn. 

Petersen R, Ryvarden L. 1971. Notes on cantharelloid fungi. IV. Two new species of Cantharellus. 
Svensk Botanisk Tidskrift 65: 399-405. 

Redhead SA, Norvell LL, Danell E, Ryman S. 2002. Proposals to conserve the names Cantharellus 
lutescens Fr.: Fr. and C. tubaeformis Fr.: Fr., Basidiomycota with conserved types. Taxon 51: 
559-562. http://dx.doi.org/10.2307/1554875 

Shao SC, Tian XE, Liu PG. 2011. Cantharellus in southwestern China: a new species and a new 
record. Mycotaxon 116: 437-446. http://dx.doi.org/10.5248/116.437 

Teng SC. 1963. Fungi of China. Science Press, Beijing, China. 

Tian XE, Shao SC, Liu PG. 2009. Two notable species of the genus Cantharellus Adans (Cantharellales, 
Basidiomycota) new to China. Edible Fungi of China 28(4):10-11 

Zang M. 1980. Some new species of Basidiomycetes from the Xizang autonomous region of China. 
Acta Microbiologica Sinica 20: 29-34. 

Zhuang WY. 2001. Higher fungi of tropical China. Mycotaxon Ltd., Ithaca, New York. 

Zhuang WY. 2005. Fungi of northwestern China. Mycotaxon Ltd., Ithaca, New York. 


ISSN (print) 0093-4666 © 2012. Mycotaxon, Ltd. ISSN (online) 2154-8889 


MY COTAXON 


http://dx.doi.org/10.5248/120.105 
Volume 120, pp. 105-118 April-June 2012 


Seven lichen species new to Poland 


MARTIN KuKWA"*, ANNA LUBEK’, RAFAEL SZYMCZYK? & ANNA ZALEWSKA‘* 


"Department of Plant Taxonomy and Nature Conservation, University of Gdansk, 
Al. Legionéw 9, PL-80-441 Gdansk, Poland 
?Jan Kochanowski University, Institute of Biology, Swietokrzyska 15A, PL-15-406 Kielce, Poland 
* EKOPROJEKT Environmental Survey Laboratory, Nowica 24, PL-14-405 Wilczeta, Poland 
* Department of Botany and Nature Protection, University of Warmia and Mazury in Olsztyn, 
pl. Lodzki 1, PL-10-727 Olsztyn, Poland 
*CORRESPONDENCE TO: dokmak@ug.edu.pl 


ABSTRACT — Biatora pontica, Buellia violaceofusca, Catillaria croatica, Parmelia ernstiae, 
Placopsis lambii, Protoparmelia oleagina and Scoliciosporum curvatum are recorded as new 
to Poland. Most reported species represent a group of crustose, often sterile lichens where 
secondary chemistry plays an important role in the taxonomy. Characteristics of each species, 
notes on similar taxa, distribution, and habitat are provided. 


KEY worps — lichenized fungi, chemotaxonomy, lichen metabolites 


Introduction 

Since the publication of the list of lichens and lichenicolous fungi of Poland 
(Faltynowicz 2003), numerous species have been added to the lichen biota of 
the country, including some new to science (e.g. Flakus 2007, Kukwa & Kubiak 
2007, Motiejunaité & Czyzewska 2008, Krzewicka 2009, Kukwa & Flakus 2009, 
Lubek 2009, Zhurbenko et al. 2009, Sliwa & Flakus 2011). Most of these taxa 
are either lichenicolous fungi or lichens belonging to critical groups requiring 
analyses of secondary lichen metabolites. 

In this paper, we report seven lichen species not previously reported from 
Poland. 


Material & methods 

The material studied is deposited in BILAS, KTC, OLS, PRA, UGDA, UPS, and herb. 
Printzen. Lichen substances were studied by thin-layer chromatography (TLC) using 
the methods of Culberson & Kristinsson (1970) and Orange et al. (2001). The examined 
localities are presented according to the modified ATPOL grid square system (Cieslinski 
& Faltynowicz 1993, Kukwa et al. 2002). 


106 ... Kukwa & al. 


The species 


Biatora pontica Printzen & Tonsberg, Biblioth. Lichenol. 86: 140. 2003. 

CHARACTERISTICS — ‘The species is characterized by its thin, crustose and 
areolate thallus with punctiform to confluent, yellowish or light green soralia; 
areoles in non-sorediate parts reaching < 1.2 mm in diam. Apothecia are 
flat to moderately convex, dark greyish-ochre to brownish-grey and 0.5-0.7 
mm in diam.; sometimes they are immersed between soralia. Anatomically 
apothecia are characterized by the presence of a blue pigment (Pontica- 
blue) in the hypothecium, subhymenium, and (rarely) hymenium and 
epihymenium; sometimes an additional red pigment (Pontica-red) is produced 
in the hypothecium and hymenium. Ascospores are ellipsoid to bacilliform, 
11.9-14.8 x 3.4-3.7 um. Biatora pontica produces thiophanic acid (major), 
asemone, and an unidentified diagnostic substance called ‘pontica unknown, 
sometimes with minor or trace amounts of other xanthones; soralia react C+ 
orange (Printzen & Tonsberg 2003). In all Polish specimens thiophanic acid, 
asemone, and ‘pontica unknown’ were detected. In only one specimen were 
apothecia developed, but the fertile material is only the second such record in 
Europe (Printzen & Tonsberg 2003). 

ECOLOGY & DISTRIBUTION — Previously B. pontica has been known from 
scattered localities in Europe (Austria, Italy, Norway, Russia, Slovenia), Asia 
(Turkey), and North America (USA) (Printzen & Tonsberg 2003, Santesson 
et al. 2004, Urbanavichus 2010). It is an epiphytic lichen growing in forests on 
deciduous trees in Europe and in Turkey, but in eastern North America it is 
more commonly found on Picea orientalis (Printzen & Tonsberg 2003). 

In Poland B. pontica has been recorded in three localities in northern Poland, 
being always found on the bark of Carpinus betulus and Fagus sylvatica in the 
bottom of shady river or streams valleys. 

SPECIMENS EXAMINED — POLAND. DRAwSKIE LAKELAND. Drawienski National 
Park, Stara Wegornia range in valley of the Plociczna river, c. 2 km NNE Ostrowite 
village, 53°04’42-46"N 15°59'31-41”E, ATPOL grid square Cb-22, on Carpinus betulus, 
08.05.2010, M. Kukwa 7948 (UGDA); ELBLAsSKA UPLAND. Valley of Srebrny Potok 
stream, forest section No. 342, 54°10'28"N 19°27'55"E, ATPOL grid square Bd-06, 
beech forest in the bottom of the valley, on Fagus sylvatica, 05.08.2007, R. Szymczyk 
641 (OLS L-691, dupl. in herb. Printzen); valley of Grabianka river, forest section No. 
183b, 54°16'42"N 19°31'27"E, ATPOL grid square Ad-96, beech forest, bottom of the 
river valley, on Fagus sylvatica, 27.06.2009, R. Szymezyk 657 (OLS L-762, dupl. in herb. 
Printzen). 
ComMENTs — When fertile, B. pontica can be confused with B. britannica 
Printzen et al. (sorediate), B. hypophaea Printzen & Tonsberg (esorediate), 
and B. ocelliformis (Nyl.) Arnold (esorediate) —three other members of the 
genus with grey apothecia. They can be easily separated chemically as they 
produce argopsin (thalli react P+ orange-red) (Printzen et al. 2001, Printzen 


Seven lichens new to Poland ... 107 


& Tonsberg 2003); in Poland only B. ocelliformis has been recorded so far 
(Faltynowicz 2003). Sterile specimens of B. pontica are morphologically very 
similar to B. efflorescens (Hedl.) Rasanen, which, however, contains argopsin 
(Printzen et al. 2001). Biatora chrysantha (Zahlbr.) Printzen and Mycobilimbia 
epixanthoides (Nyl.) Vitik. et al. can also be confused with B. pontica, but they 
contain gyrophoric acid (B. chrysantha) or no lichen metabolites are produced 
(M. epixanthoides) (Tonsberg 1992, Printzen & Tonsberg 2003, Printzen & Otte 
2005). 

Biatora pontica can also be confused with Pyrrhospora quernea (Dicks.) 
Korb. which is morphologically very similar when sterile (greenish sorediate 
thallus). Both taxa contain thiophanic acid as the major secondary metabolite, 
but P. quernea lacks the characteristic substance ‘pontica unknown’ (Tonsberg 
1992, Printzen & Tonsberg 2003). 


Buellia violaceofusca G. Thor & Muhr, Lichenologist 23: 11. 1991. 

CHARACTERISTICS — ‘The species is characterized by its thin, usually 
endophloeodal, pale grey or almost white thallus producing dark brownish 
soralia with a violet tinge (but greenish when abraded). Soralia are maculiform, 
scattered, slightly elevated and usually confluent; no apothecia have been 
discovered so far. This lichen does not produce secondary metabolites (Thor & 
Muhr 1991), but traces of terpenoids from tree bark can be detected by TLC. 

ECOLOGY & DISTRIBUTION — Buellia violaceofusca is widespread but with 
a scattered distribution (Coppins et al. 2009); so far it has been found only 
in Europe in Austria (Poelt 1994), Belgium (Sérusiaux et al. 1999), Estonia 
(Thor & Nordin 1998), Great Britain (Coppins et al. 2009), Norway (Gaarder & 
Tonsberg 2010), Slovakia (Palice et al. 2006), and Sweden (Thor & Muhr 1991). 
The species usually grows on bark of Quercus spp. in different types of forest, 
but in Great Britain it has been reported also on Fraxinus excelsior (Coppins et 
al. 2009). 

In Poland it has been found only at one locality in bark crevices of old Quercus 
sp. in a deciduous forest. Most probably the species is much more common in 
the country but has been overlooked due to the inconspicuous thalli and the 
habit of growing in deep crevices. 

SPECIMEN EXAMINED — POLAND. KASZUBSKIE LAKELAND. Trdjmiejski Landscape 
Park, Dolina Ewy valley, 54°24'35"N 18°30'20"E, ATPOL grid square Ac-89, oak- 
hornbeam forest, on Quercus sp., 04.04.2004, M. Kukwa 3006 (UGDA L-10664). 
REFERENCE MATERIAL EXAMINED — SWEDEN. VARMLAND. VISNUM KIL PAR., 
N6t6n nature reserve, Arskagen, near Lake Vaner, 59°04'N 14°01’E, 50 m, on the 
northern side of old Quercus robur in deciduous wood, 27.05.1985, L.-E. Muhr 7911 
(UPS L-17315 — holotype). 
ComMENTS — Buellia violaceofusca is a very characteristic epiphytic species due 
to its thin thallus, violet tinged soredia, and the absence of lichen metabolites. 


108 ... Kukwa & al. 


In Poland it can be confused with the morphologically similar B. griseovirens 
(Turner & Borrer ex Sm.) Almb. Both species have dark outermost soredia, but 
B. griseovirens has a thicker thallus and produces atranorin and norstictic acid, 
with trace of connorstictic acid and sometimes unidentified pigments (in some 
specimens only atranorin or only norstictic acid are present) (Thor & Muhr 
1991, Tonsberg 1992, Coppins et al. 2009). 

Buellia arborea Coppins & Tonsberg also develops pigmented soredia, but 
it can be readily separated by the presence of atranorin and placodiolic acid 
(Tonsberg 1992, Coppins et al. 2009); so far the species has not been reported 
from Poland, but its discovery there is probable. 


Catillaria croatica Zahlbr., Ann. Mycol. 4: 487. 1906. 
= Lecania croatica (Zahlbr.). Kotlov, Novosti Sist. Nizsh. Rast. 37: 251. 2004. 

CHARACTERISTICS — As the only modern description of C. croatica (by 
Harris & Lendemer 2010) is based mainly on North American specimens, a 
more detailed description is provided below. The thallus of this corticolous 
lichen is light green-grey, superficial, well or poorly developed to immersed, 
consisting of scattered or almost contiguous areoles. Soralia are numerous, 
rounded, flat, convex or weakly capitate, discrete or patchily coalescing to 
form a leprose crust. Soredia are in shades of green, but sometimes externally 
pale brown pigmented. Apothecia are sessile, flat or weakly convex, with pale 
orange-cream or medium brown discs and paler margins. Anatomically, all 
structures are colourless except the pale brownish hypothecium. Asci are 
8-spored with narrowly fusiform, 0-1-septate ascospores measuring 12-17 
x 4.5 um. Pycnidia were not found in Polish material. The species does not 
produce lichen substances detectable by TLC. 

ECOLOGY & DISTRIBUTION — Rarely reported from Europe: Austria, 
Belgium, Croatia, France, Luxembourg, Romania, Slovakia, Slovenia and 
Ukraine (Printzen 1995, Mrak et al. 2004, Hafellner et al. 2005, Eichler et al. 
2010, Vondrak et al. 2010), and also known from North America (Harris & 
Lendemer 2010). The species grows in forests, usually on bark of broad-leaved 
trees, rarely also shrubs (e.g. Sambucus sp.). 

In Poland, C. croatica has been found in only one locality, a well-preserved, 
humid deciduous forest not seriously influenced by forest management, growing 
in association with Anisomeridium polypori (Ellis & Everh.) M.E. Barr, Biatora 
ocelliformis, Lepraria lobificans Nyl., L. vouauxii (Hue) R.C. Harris, Opegrapha 
rufescens Pers., Pertusaria pertusa (L.) Tuck., and Bacidia sp. 

SPECIMEN EXAMINED — POLAND. BIELSK PLAIN. Bialowieski National Park, forest 
section No. 273, 52°43°32”N, 23°53’27°E, ATPOL grid square Cg-46, on Acer sp., 
27.06.2009, A. Lubek s.n. (KTC). 
ComMMENTS — Catillaria croatica is an inconspicuous, easily overlooked, 
corticolous lichen with a sorediate thallus lacking lichen metabolites, with small 


Seven lichens new to Poland ... 109 


pale apothecia and 0-1-septate ascospores. The species is morphologically very 
similar to Mycobilimbia epixanthoides: both are sorediate, lack lichen substances, 
and produce pale biatorine apothecia. When fertile, the material is much easier 
to determine as both species differ in the type of spores: C. croatica produces 
0-1-septate ascospores, whereas M. epixanthoides has 3-septate ascospores. In 
the sterile state they may be separated based on substrate preference: Catillaria 
croatica grows exclusively on the bark of trees, and M. epixanthoides prefers 
bryophytes and is rarely corticolous (Hafellner et al. 2005, Harris & Lendemer 
2010). The corticolous material of the latter, according to the cited sources, 
could be distinguished by irregular young soralia forming larger irregular 
patches, while soralia of C. croatica remain discrete and round, even when they 
become crowded with age. From our experience, this might not be true in all 
cases and some sterile samples may remain undetermined. 

Several other sorediate species, which are morphologically similar to 
C. croatica (e.g. Biatora efflorescens or B. chrysantha), can be confused with this 
lichen, but they are all readily distinguished by their secondary metabolites 
(e.g., Printzen 1995). 

Catillaria croatica has been recently transferred to Lecania A. Massal. by 
Kotlov (2004), but molecular studies have shown that it nests in Bilimbia De 
Not. s.l. and is not phylogenetically related to Lecanias. str. (Reese Neesborg et al. 
2007). Therefore we prefer to keep the species in the admittedly heterogeneous 
genus Catillaria A. Massal. 


Parmelia ernstiae Feuerer & A. Thell, Mitt. Inst. Allg. Bot. Hamburg 30-32: 52. 2002. 

CHARACTERISTICS — This foliose lichen is characterized by its heavily 
pruinose thalli and isidia, small and non-overlapping lobes, commonly 
present lobulae, and the production of atranorin (minor amount, together 
with chloratranorin), salazinic acid (major amount, with minor amount of 
consalazinic acid), lobaric, lichesterinic, protolichesterinic, nephrosteranic, 
isonephrosterinic (all minor amounts) and protocetraric (trace amount) acids 
(Feuerer & Thell 2002, Thell et al. 2008). Although not distinctly lobulate, 
Polish material agrees well with the description, except nephrosteranic, 
isonephrosterinic and protocetraric acids were not detected, most probably due 
to their low quantity or overlapping with other metabolites in a similar position 
on the TLC plates. 

ECOLOGY & DISTRIBUTION — Parmelia ernstiae is widely distributed in 
Europe, with a few records known from Africa. It has been reported from 
Austria, Belgium, Bosnia and Herzegovina, Britain, Bulgaria, Czech Republic, 
Denmark, Estonia, France, Germany, Greece, Ireland, Lithuania, Luxembourg, 
the Netherlands, Slovenia and Spain (Feuerer & Thell 2002, Sérusiaux et al. 
2003, Thell 2003, Molina et al. 2004, Otte 2005, Santesson et al. 2004, Thell 


110 ... Kukwa & al. 


et al. 2007, Suija et a. 2007, Motiejiinaité et al. 2008, Thell et al. 2008, Berger 
et al. 2009, Seaward 2010, Diederich et al. 2011) and in Africa from Algeria 
and the Canary Islands (Sérusiaux et al. 2003). It is a typically corticolous 
species growing on deciduous or rarely coniferous trees in open situations (e.g. 
roadsides, churchyards) and forests (Thell 2003, Santesson et al. 2004, Thell et 
al. 2007); an exceptional saxicolous record is also known (Thell et al. 2008). 

In Poland it has been found only at one locality in northwestern Poland ona 
roadside elm in a well-lit situation. All available material labelled as P. saxatilis 
in UGDA has been examined, but as no more specimens of P. ernstiae have 
been traced, it probably should be regarded as rare in the country. 

SPECIMEN EXAMINED — POLAND. DRAwSKIE LAKELAND. road N of Gtusko village, 

53°02'59"N 15°56'34"E, ATPOL grid square Cb-22, on roadside Ulmus sp., 05.05.2010, 

M. Kukwa 7793 (UGDA). 
CoMMENTS — Based on DNA sequences and different morphology (Feuerer 
& Thell 2002) and more recently on the composition of secondary metabolites 
(Thell et al. 2008), P ernstiae has been recently segregated from P. saxatilis (L.) 
Ach., from which it can be easily separated morphologically by the epruinose 
thalli and isidia and chemically by the absence of lobaric and fatty acids (Feuerer 
& Thell 2002, Thell et al. 2008). 

Parmelia ernstiae can also be confused with another isidiate species, 
P. serrana A. Crespo et al. Thalli of both taxa are pruinose, but in P serrana 
lobes are larger, rounded, and commonly overlapping and lobaric acid is not 
produced (Molina et al. 2004, Thell et al. 2008). Parmelia serrana has not been 
found in Poland, although its distribution range (see Thell et al. 2008) suggests 
it probably occurs there. 


Placopsis lambii Hertel & V. Wirth, Flecht. Baden-Wiirtt. Verbreit.: 511. 1987. 
CHARACTERISTICS — ‘The species is characterized by its placodioid thallus 
with deeply incised and radiating marginal lobes, shiny upper surface, usually 
blackish and more or less rounded soralia, non-lobate cephalodia (absent in 
some specimens), and the production of 5-O-methylhiascic and gyrophoric 
acids as major substances (Moberg & Carlin 1996, Gilbert & Purvis 2009, 
Harrold et al. 2010). The cephalodia were not developed in Polish specimen. 
ECOLOGY & DISTRIBUTION — Placopsis lambii is widespread but rather 
scattered. In Europe it has been reported from Austria, Belgium, Britain 
Finland, France, Germany, Iceland, Ireland, the Netherlands, Norway, Russia, 
and Sweden (Wirth 1987, Diederich 1994, Moberg & Carlin 1996, Aptroot et al. 
1999, Hafellner & Turk 2001, Santesson et al. 2004, Harrold et al. 2010, Seaward 
2010, Diederich et al. 2011). In Africa it has been found in Kenya, Lesotho, and 
Tanzania (Moberg & Carlin 1999) and in the Americas in Bolivia, Chile, Costa 
Rica, and Ecuador (Galloway 2002, Galloway & Arvidsson 2007). It is also 


Seven lichens new to Poland... 111 


known from New Zealand (Galloway 2001) and asiatic Russia (eastern Siberia 
and Russian Far East; Urbanavichus 2010). 

In Poland P. lambii has been recorded only once in northern Poland where it 
was found on a pile of stones in a sunny but rather humid habitat near several 
ponds. This stand is also noteworthy for other rare and endangered lichens in 
Poland, e.g., Rhizocarpon lecanorinum Anders and Xanthoparmelia mougeotii 
(Schaer.) Hale. 

SPECIMEN EXAMINED — POLAND. KaASZzuBSKIE LAKELAND. Vicinity of Szumles 

village, 54°09'33"N 18°13'26"E, ATPOL grid square Bc-07, open area by small ponds, 

on stone, 08.05.2008, M. Kukwa 5968, M. Bulinski, J. Zarembska (UGDA L-16515, dupl. 

in BILAS and PRA). 
ComMENTsS — Placopsis lambii is very similar and morphologically almost 
indistinguishable from P gelida (L.) Linds. Placopsis gelida tends to be matte or 
shiny only at the lobe tips of the upper surface and has more elongated soralia that 
are rarely blackish. However, those are subjective characters, and the chemistry 
is a most reliable diagnostic feature; both taxa contain gyrophoric acid, but 
P. lambii additionally produces 5-O-methylhiascic acid (Gilbert & Purvis 2009, 
Harrold et al. 2010). Due to their similarity one could treat both species as one 
variable entity, but molecular studies supported their distinctiveness (Schmitt 
et al. 2003). 

Some P. lambii morphs might be also mistaken for Trapelia placodioides 
Coppins & P. James, which, however, lacks radiating marginal lobes and 
cephalodia; T’ placodioides also produces gyrophoricacid, but 5-O-methylhiascic 
acid is absent (Gilbert & Purvis 2009, Purvis et al. 2009). 


Protoparmelia oleagina (Harm.) Coppins, Lichenologist 24: 368. 1992. 
= Lecanora oleagina Harm., Lich. France 5: 1023. 1913. 

CHARACTERISTICS — Protoparmelia oleagina is a crustose, often sterile, 
epiphytic lichen with a more or less continuous, dull olivaceous brown, scurfy 
granular-isidiate thallus and apothecial margin; the apothecial disc is also 
olivaceous. Ascospores are fusiform, 9.5-15 x 2-3.5 um (Coppins & Chambers 
2009, Brodo & Aptroot 2005). The species produces lobaric acid, but often in 
low concentration (Brodo & Aptroot 2005). One Polish specimen was fertile 
(Kukwa 7837a), but very few apothecia were developed. 

ECOLOGY & DISTRIBUTION — Protoparmelia oleagina occurs only in Europe, 
where it has been recorded from Austria, Belgium, Finland, France, Germany, 
Great Britain, Italy, the Netherlands, Norway and Sweden (Aptroot et al. 1999, 
Scholz 2000, Hafellner & Turk 2001, Santesson et al. 2004, Brodo & Aptroot 
2005, Nimis & Martellos 2008, Coppins & Chambers 2009, Diederich et al. 
2011). 

In Poland P. oleagina has been found on deciduous trees in humid forests at 
two localities in western Pomerania (NE Poland). 


112 ... Kukwa & al. 


SPECIMENS EXAMINED — POLAND. DRAWSKIE LAKELAND. Drawienski National Park, 

eastern slopes of Drawa river valley, 2 km NE of Zatom village, Tragankowe Urwisko 

range, 53°09'12"N_ 15°51'49-54"E ATPOL grid square Cb-12, deciduous forest, on 

Fagus sylvatica, 06.05.2010, M. Kukwa 7837a (UGDA). SLOWINSKIE Coast. Bialogora 

nature reserve, forest section No. 22, 54°49'27"N, 17°57'52"E, ATPOL grid square Ac- 

36, wet pine-birch forest, on Betula pendula, 22.09.2010, M. Kukwa 8268a, A. Jabtonska, 

M. Oset (UGDA L-16660). 
ComMENTS — Protoparmelia oleagina is easily distinguished by its corticolous 
habitat and more or less continuous, dull olivaceous brown, scurfy granular 
and isidiate thallus. Morphologically it is most similar to P. ochrococca (Nyl.) 
P.M. Jorg. et al., but the thallus of the latter is not isidiate, consists of subglobose 
areoles, and lacks lichen substances (Brodo & Aptroot 2005, Coppins & 
Chambers 2009); so far P. ochrococca has not been recorded from Poland. 

Chemically and morphologically P oleagina can be confused with 
P. hypotremella Herk et al., which is known from several localities in Poland 
(see Kubiak et al. 2010). Both taxa contain lobaric acid, but the thallus of 
P. hypotremella consists of dispersed round flat granules or microsquamules; 
it is also brighter, pale grayish-brown to olive-grey (Brodo & Aptroot 2005, 
Coppins & Chambers 2009). 

Morphologically, P oleagina resembles small forms of P. badia (Hoftm.) 
Hafellner, but the latter is a saxicolous lichen with ellipsoid-fusiform ascospores 
with distinctly pointed apices (Coppins & Chambers 2009). 

Some epiphytic species of the genus Lecanora Ach. also have dark apothecial 
discs (e.g. L. argentata (Ach.) Malme or L. persimilis (Th. Fr.) Arnold), but they 
lack brown pigmentation, often produce atranorin or usnic acid in the cortex, 
and their ascospores are generally wider (Sliwa 2007, Coppins & Chambers 
2009). 


Scoliciosporum curvatum Sérus., Nord. J. Bot. 13: 458. 1993. 

CHARACTERISTICS — This crustose lichen is characterized by its pale green 
to dark grey-green granular thallus, chlorococcoid photobiont with rather large 
cells (12—)14—20(-22) um, numerous and very small (up to 0.16 mm diam.), 
pale pink, orange to brown apothecia, 2 um wide, simple to furcated or rarely 
anastomosing paraphyses, 8-16-spored asci and 1-septate, curved (lunulate) 
or slightly sigmoid spores with acute ends, 7-11 x 1.5-3 um. Paraphyses 
are abundant only in young apothecia. The species does not produce lichen 
substances (Sérusiaux 1993, Edwards et al. 2009). 

ECOLOGY & DISTRIBUTION — This species occurs only in Europe, being much 
more common in western countries. It has been reported from Austria, Britain, 
Czech Republic, Denmark, France, Germany, Hungary, Ireland, Norway, 
Slovakia, Romania, Spain, Sweden, Switzerland and Ukraine (Sérusiaux 1993, 
Poelt 1994, Boom et al 1995, Palice 1999, Sochting & Alstrup 2002, Clerc 2004, 


Seven lichens new to Poland... 113 


Santesson et al. 2004, Lisickaé 2005, Edwards et al. 2009, Vondrak et al. 2009, 
Seaward 2010, Dymytrova 2011). Dymytrova (2011), citing Nimis & Martellos 
(2003), cited S. curvatum from Italy, but Nimis & Martellos (2008) have included 
it not in the Italian checklist but only in the iconographic archive (along with 
several other species not reported from Italy). The report by Himelbrant (2008) 
of S. curvatum from Poland is a misprint (Himelbrant, pers. comm.), making 
the specimens cited below the first records for Poland. 

Most central European localities of S. curvatum lie in the mountains 
(490-1600 m a.s.l.), but those from Poland are in the lowlands. The species is 
treated as an endangered species (extremely rare and potentially endangered 
or vulnerable) in some countries and has been included in the lichen red lists 
of Austria (Turk & Hafellner 1999), Czech Republic (Liska et al. 2008), and 
Switzerland (Scheidegger et al. 2002). 

Scoliciosporum curvatum is typically a foliicolous lichen growing in sheltered 
and humid situations on leaves and (sometimes) twigs of evergreen shrubs 
such as Buxus, Camellia, and Rhododendron (Edwards et al. 2009). The species 
has also been found on needles (rarely twigs) of spruces, firs and occasionally 
pines, typical of southwestern Scandinavia (Santesson et al. 2004) and the 
mountains of central Europe (Poelt 1994, Palice 1999, Guttova & Palice 1999, 
Lisicka 2005). 

In Poland S. curvatum has been found only in two localities in Romincka 
Forest in northeast Poland where it was collected from spruce needles inside a 
wet spruce forest and in a managed pine forest. 

SPECIMENS EXAMINED — POLAND. RoMINCKA Foresst. Puszcza Romincka Forest 
Landscape Park, Zytkiejmska Struga nature reserve, forest section No. 63, c. 3.5 km 
WSW of Zytkiejmy village, 54°20'51"N 22°37'55"E, ATPOL grid square Af-86, boggy 
spruce forest, on needles of spruce, 17.09.2003, A. Zalewska, W. Faltynowicz s.n. (OLS); 
forest section No. 184, c. 4.5 km of SW Zytkiejmy village, 54°19'49"N 22°37'37"E, 
ATPOL grid square Af-86, pine forest with spruce, on needles of spruce, 19.09.2003, A. 
Zalewska, W. Faltynowicz s.n. (OLS). 
CoMMENTS — Placement of S. curvatum within Scoliciosporum A. Massal. is 
problematic as it is separated from other representatives by its higher number 
of ascospores per ascus and different organization of paraphyses (Sérusiaux 
1989, 1993). The generic concept appears to be unclear, since several other 
species with deviating characters are included, especially S. abietinum 
T. Sprib., S. intrusum (Th. Fr.) Hafellner, and S. coniectum Kantvilas & Lumbsch 
(Sérusiaux 1993, Hafellner 2004, Spribille et al. 2009, Kantvilas & Lumbsch 
2010). Molecular analyses would clarify its circumscription and explain its 
relationship with S. curvatum. 

Scoliciosporum curvatum is inconspicuous and easily overlooked, as 
its thallus may resemble a green algal cover over the substrate. In the field 
it can be confused with other species growing in the same habitat, but it is 


114... Kukwa & al. 


easily recognizable after microscopic examination. In Poland, only two other 
species, Fellhanera bouteillei (Desm.) Vézda and F. subtilis (Vézda) Diederich 
& Sérus., have been found on needles of Abies alba (F. bouteillei) or Picea abies 
(FE. subtilis) (Faltynowicz 2003). The thallus of E subtilis is grey to green with 
smaller photobiont cells (5-15 um in diam.), apothecia are whitish and larger 
(0.15-0.4 mm in diam.), ascospores are oblong, fusiform and (1-)3-septate, 
and it always develops pinkish pycnidia. Fellhanera bouteillei has a verruculose- 
rimose thallus, often entirely covered by dull bluish-green farinose soredia. 
The apothecia and pycnidia superficially resemble those of F subtilis, but 
ascospores are only 1-septate (as in S. curvatum) but also ovoid to oblong-ovoid 
(sometimes soleiform) and often constricted at the septa; the F. bouteillei thallus 
also contains usnic acid, zeorin, and + asemone (Sérusiaux 1996, Aptroot et al. 
2009). 

Some S. curvatum specimens might also be mistaken for Fellhanera 
viridisorediata Aptroot et al., which has not been found in Poland. It has a 
similar green granular thallus, with firstly punctiform, then coalescing, soralia 
and (0—)1-septate elongate-ellipsoid ascospores. The species is often sterile, but 
then it differs in the presence of roccellic acid (Aptroot et al. 2009). 

Scoliciosporum gallurae Vézda & Poelt, recently recorded in Poland 
(Kukwa & Kubiak 2007), which may also superficially resemble S. curvatum, 
produces gyrophoric acid and is known from twigs in open, nutrient-enriched 
situations. 


Acknowledgments 

We are very grateful to Dr Jurga Motiejunaité (Vilnius) and Prof. Mark R. D. Seaward 
(Bradford) for reviewing the manuscript, and very valuable comments on a previous 
version of the paper. We would also like to thank Dr Christian Printzen (Frankfurt am 
Main) and Dr Zdenék Palice (Prihonice) respectively for confirming our determinations 
of Biatora pontica and Placopsis lambii, Dr Anders Nordin (Uppsala) for hospitality 
during visits to UPS, and Dr Dmitry E. Himelbrandt (Saint Petersburg) for information 
on the distribution of Scoliciosporum curvatum. 


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ISSN (print) 0093-4666 © 2012. Mycotaxon, Ltd. ISSN (online) 2154-8889 


MY COTAXON 


http://dx.doi.org/10.5248/120.119 
Volume 120, pp. 119-125 April-June 2012 


Dissoconium proteae newly recorded from China 


RONG ZHANG’, YONGNA Mao’, Lu Hao’, 
HONGCAI CHEN?, GUANGYU SUN’ & MARK L. GLEASON? 


' State Key Laboratory of Crop Stress Biology in Arid Areas and College of Plant Protection 

& College of Life Sciences, Northwest A&F University, Yangling, Shaanxi 712100, P. R. China 
*Department of Plant Pathology and Microbiology, Iowa State University, Ames, Iowa 50011, U.S.A 
* CORRESPONDENCE TO: sgy@nwsuaf.edu.cn 


ABSTRACT — We document the first report of Dissoconium proteae from China. This fungus 
was found on the cuticle of apple fruit collected from orchards in Shaanxi Province. Its 
morphology is compared with other Dissoconium species and a phylogenetic analysis based 
on ITS sequence is presented. 


Key worps — sooty blotch and flyspeck, taxonomy, internal transcribed spacer, rDNA 


Introduction 

Sooty blotch and flyspeck (SBFS) is a worldwide disease complex more 
than 60 fungi that blemish the cuticle of pome fruits (such as apple) in humid 
temperate regions and cause cosmetic damage entailing significant economic 
losses (Batzer et al. 2005). The common name “flyspeck” refers to species in the 
SBFS complex that appear on fruit surfaces as clusters of black, shiny, round 
to oval, flattened sclerotium-like bodies with no mycelial mat (Batzer et al. 
2005). 

The hyphomycete genus Dissoconium de Hoog et al. has lightly pigmented 
conidiophores that produce septate and aseptate conidia in a sympodial manner. 
The conidia are hyaline, one- or two-celled, and detach forcibly in pairs (Hoog 
et al. 1983). 

Currently the genus includes nine species: Dissoconium aciculare de Hoog 
et al., D. australiense Crous & Summerell, D. commune Crous & Mansilla, 
D. dekkeri de Hoog & Hijwegen, D. eucalypti Crous & Carnegie, D. maliG.Y. Sun 
et al., D. musae Arzanlou & Crous, D. proteae, and D. subuliphorum (Matsush.) 
R.F. Castaneda (Arzanlou et al. 2008; Crous et al. 1999, 2004, 2007, 2008; Hoog 
et al. 1983, 1991; Zhang et al. 2007). Batzer et al. (2005) reported five putative 
Dissoconium species within the sooty blotch complex on apple fruit. 


120 ... Zhang & al. 


Recently, during investigations of SBFS of apple in China, we identified four 
fungal isolates that represent the first record of Dissoconium proteae from China 
based on ITS sequence analysis and morphological comparison. 


Materials & methods 

IsOLATION. In September 2008, apples with flyspeck signs were collected from 
orchards in Shaanxi Province. The thalli were transferred to a potato dextrose agar slant 
(potato 200 g, dextrose 20 g, agar 10 g; PDA) directly from the apple surface, and then 
cultured at 23+1°C in darkness (Sun et al. 2003). Colony description of the species was 
based on a 1-month-old pure culture. Morphological characteristics are described from 
specimens obtained from a cover slip inserted into the surface of the media. Voucher 
specimens and cultures are conserved in the Fungal Herbarium of Northwest A&F 
University, Yangling, China (HMUABO). 

DNA SEQUENCING. Template DNA was extracted from fungal mycelium according 
to Li et al. (2011); primer pairs used for ITS amplification and sequencing were ITS1-F 


TABLE 1. Sequences used in the phylogenetic analysis 


SPECIES GENBANK CODE REFERENCE 

Dissoconium proteae (mynbxd10) HM070373 This paper 

D. proteae (mynbxd2) HM070374 This paper 

D. proteae (mynbxd1.1) HM070375 This paper 

D. proteae (mynxc1.1) HM070376 This paper 

D. aciculare AY725519 Crous et al. (2004) 
AY725521 Crous et al. (2004) 

D. australiense EF394854 Crous et al. (2007) 

D. eucalypti EF394855 Crous et al. (2007) 

D. mali EF627451 Zhang et al. (2007) 

D. musae EU514226 Arzanlou et al. (2008) 
EU514225 Arzanlou et al. (2008) 

D. proteae EU707897 Crous et al. (2008) 

Dissoconium sp. AY598874 Batzer et al. (2005) 
AY598875 Batzer et al. (2005) 
AY598877 Batzer et al. (2005) 
FJ425205 Diaz Arias et al. (2010) 


Mycosphaerella communis 


(Gtisni. Deeorintune) AY725544 Crous et al. (2004) 

DQ302950 Crous et al. (2006) 

M. lateralis (anam.: D. dekkeri) EU301078 Burgess et al. (2007) 

Ramichloridium apiculatum EU041794 Arzanlou et al .(2007) 
EU041791 Arzanlou et al .(2007) 

R. cerophilum EU041798 Arzanlou et al. (2007) 

R. indicum EU041799 Arzanlou et al .(2007) 

Ramichloridium sp. FJ425199 Diaz Arias et al. (2010) 


Stenella araguata AF362066 Crous et al. (2001) 


Dissoconium proteae new to China... 121 


(Gardes & Bruns 1993) and ITS4 (White et al. 1990). Amplification was completed with 
the following cycling parameters: initial denaturation at 94°C for 3 min followed by 35 
cycles of denaturation at 94°C for 30 s, annealing at 52°C for 30 s, and extension at 72°C 
for 10 min. The PCR products were sequenced by Organism Technology Co., Shanghai, 
China. 

The ITS nucleotide sequences generated in this study were added to sequences 
downloaded from GenBank (TABLE 1) that had high similarity according to a BLAST 
search (National Center for Biotechnology Information’s nucleotide blast program). 
Preliminary alignments were performed using CLUSTAL-X (1.83) (Thompson et 
al. 1997), imported into BioEdit version 5.0.9.1 (Hall 1999), and manually adjusted. 
Phylogenetic analysis of aligned DNA sequences was performed with PAUP version 
4.0b10 for 32-bit Microsoft Windows (Swofford 2001). Heuristic searches were 
performed with 1000 random sequence additions. Clade stability was evaluated by 1000 
bootstrap replications. Other measures for parsimony, including tree length, consistency 
index, retention index and rescaled consistency index (CI, RI and RC, respectively), 
were also calculated. Stenella araguata was used as the outgroup taxon. 


Results: DNA phylogeny 

A multiple alignment of the rDNA-ITS was generated with 21 sequences 
obtained from GenBank plus the sequences of isolates mynbxd10, mynbxd2, 
mynbxd1l.1 and mynxcl.l. An MP tree with 354 length (CI = 0.7232, 
RI = 0.8622, RC = 0.6235) was constructed (Fic. 1). Two major clades were 
resolved in the MP trees. One of the major clade with 100% bootstrap value 
contained three species in Dissoconium, Mycosphaerella, and Ramichloridium. 
The other major clade also had a bootstrap value of 100%. Our isolates and 
an authentic Dissoconium proteae isolate identified by Crous et al. (2008) fell 
within a single clade with 71% bootstrap support. 


Taxonomy 


Dissoconium proteae Crous, Persoonia 20: 68. 2008. Fic. 2 

Mycelium external consisting of branched, septate, smooth, hyaline hyphae, 
0.8-2.8 um wide. Conidiophores 9-20 x 2.2-4.7 um, solitary, arising from 
hyphae, subcylindrical, subulate, tapering to a bluntly rounded or truncate 
apex, straight to gently curved, smooth, hyaline, becoming medium pale brown 
with age, aseptate; conidiogenous loci terminal and lateral, visible as slightly 
thickened, darkened scars, 0.5 um wide. Conidia 6.3-11.5 x 2.2-4 um, solitary, 
hyaline to pale olivaceous, smooth, ellipsoid, non-constricted to slightly 
constricted at median septum, apex obtuse, base obconic-truncate, tapering 
pronounced at somewhat protruding hilum, unthickened, not darkened, 1 um 
wide. Secondary conidia developing adjacent to primary conidia, hyaline to 
subhyaline, aseptate, ellipsoid, tapering prominently towards a protruding, 
truncate base, 4.3-8 x 1.6-3.8 um; anastomosing with primary conidia after 


122 ... Zhang & al. 


EU301078 Mycosphaerella lateralis 
AY463143 Dissoconium aciculare 
90 || 4¥639404. Dissoconium aciculare 
89 |' EF394855 Dissoconium eucalypti 
AY598874 Dissoconium sp. 

97' AY598875 Dissoconium sp. 
EU707897 Dissoconium proteae 
mynxct.1 

mynbxd1.1 


mynbxd2 
mynbxd10 


61 


AY598877 Dissoconium sp. 
FJ425205 Dissoconium sp. 
EU041799 Ramichloridium indicum 
100, £U041794 Ramichloridium apiculatum 
EU041791 Ramichloridium apiculatum 
100 EF627451 Dissoconium mali 
EU514226 Dissoconium musae 
98 |! EU514225 Dissoconium musae 
EF394854 Dissoconium australiense 
DQ302950 Mycosphaerella communis 

91! AY725544 Mycosphaerella communis 
FJ425119 Ramichloridium sp. 
EU041798 Ramichlondium cerophilum 
AJ244261 Stenella araguata 


10_ 


Fic. 1 The majority consensus tree (length = 354, CI = 0.7232, RI = 0.8622, RC = 0.6235) derived 
from a heuristic search option in PAUP version 4.0b10 for 32-bit Microsoft Windows with 1000 
randomizations of sequence input orders and 1000 bootstrap replications using the data set of ITS1, 
5.88 and ITS2. Bootstrap values higher than 50% are indicated above or below the tree branches. 


active discharge. In some cases the secondary conidia were observed to 
germinate. 

CULTURAL CHARACTERISTICS: Colonies on PDA spreading, with sparse 
aerial mycelium and regular smooth margins; surface white or sienna, with 
patches of white and cinnamon; forming clusters of black sclerotia (remaining 
infertile) on PDA; reaching 15 mm diam after 1 mo. 

CHARACTERISTICS ON THE HOST: On apple peel, the fungus produced dark, 
shiny, round to oval, slightly protuberant sclerotium-like bodies (Fic. 2A). 

SPECIMENS EXAMINED: China: Shaanxi Province, Xianyang, Bin County, 35°04'N 
108°09’E, alt. 1108 m, on Malus xdomestica (Rosaceae), 18 Sep. 2008, Y.N. Mao, 
HMUABO (the Fungal Herbarium of Northwest A&F University) 8885, 8877, 8866 
(with dried culture), culture mynbxd10, mynbxd2, mynbxd1.1. China: Shaanxi Province, 
Xianyang, Xunyi County, 35°13’N 108°33’E, alt. 1300 m, On Malus xdomestica, 18 Sep. 
2008, Y.N. Mao, HMUABO 8682 (with dried culture), culture mynxcl.1. 


Dissoconium proteae new to China... 123 


Fic. 2 Dissoconium proteae isolate mynbxd10. A. Signs on apple peel. B. Colony on PDA 
agar after 30 days. C-D. Conidia, conidiogenous cells, and hyphae. E. Conidiogenous cell. 
F, Conidium and secondary conidium. Bars: B=10 mm, C-F =10 um. 


124 ... Zhang & al. 


Discussion 

Of the more than 60 fungal species that blemish the surface of apples 
worldwide (Diaz Arias et al. 2010), many have been described quite recently. 
For example, Li et al. (2010) and Ma et al. (2010) reported three species of 
Zygophiala (Z. cryptogama and Z. cylindrica; Z. qianensis) associated with 
flyspeck on apples. Our report provides the first evidence that Dissoconium 
proteae can cause flyspeck on apple fruit. Crous et al. (2008) described 
D. proteae from leaves of Protea sp. (Proteaceae), causing a leaf spot. 

Currently there are nine species in Dissoconium. Based on phylogenetic 
analysis of the ITS region and morphological characters of the anamorph, we 
identified four isolates as Dissoconium proteae. This is the first report of the 
species from apple, and a new record for China. 


Acknowledgments 

This work was supported by National Natural Science Foundation of China 
(31170015, 31171797), the 111 Project from Education Ministry of China (B07049), Top 
Talent Project of Northwest A&F University and the earmarked fund for Modern Agro- 
industry Technology Research System (nycytx-08-04-04).The authors wish to thank 
Dr Eric H.C. McKenzie (Landcare Research, Auckland, New Zealand) and Professor 
Zhongyi Zhang (College of Plant Protection, Yunnan Agricultural University, Kunming, 
Yunnan, China) for reviewing the manuscript. 


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Dissoconium proteae new to China... 125 


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China colonizing apples. Mycotaxon 101: 165-172. 


ISSN (print) 0093-4666 © 2012. Mycotaxon, Ltd. ISSN (online) 2154-8889 


MY COTAXON 


http://dx.doi.org/10.5248/120.127 
Volume 120, pp. 127-132 April-June 2012 


The fungal collection of the Jagiellonian University Herbarium 
(KRA), Krakow, Poland 


ANNA MarIA OCIEPA’', SZYMON ZUBEK? & PIOTR MLECZKO ” 


'W. Szafer Institute of Botany, Polish Academy of Sciences & 
?Institute of Botany, Jagiellonian University 

ul. Lubicz 46, 31-512 Krakow, Poland 
“CORRESPONDENCE TO: ubmleczk@cyf-kr.edu.pl 


ABSTRACT — ‘The paper presents a short history of the mycological collection of the 
Jagiellonian University Herbarium (KRA, Krakow, Poland). The Herbarium holds over 22,000 
specimens, including some interesting European and extra-European fungal exsiccata. One 
of the most valuable fungal collections (including many type specimens) in the Herbarium is 
that containing Javanese fungi gathered by Marian Raciborski at the end of 19" century. 


Key worps — fungi of Pieniny Mts., fungi of Gorce Mts., ascomycetes, basidiomycetes, 
Peronospora 


The history 

The origin of the Jagiellonian University Herbarium dates back to 1780, 
when the Faculty of Chemistry and Natural History was established, and with it 
the Natural History Room (Kohler 1999; Kohler 2001a). The Herbarium, which 
was long associated with the Chair of Botany at the Botanical Garden, is now 
part of the Institute of Botany of Jagiellonian University. 

The first record of fungi being kept in the Herbarium dates from 1812, and 
the oldest specimen with a date placed on the label is from 1807 (Fic. 1). In the 
past the Herbarium collections served mainly an educational function (Kohler 
2001b). Until 1910 each set of specimens, including purchased, exchanged, or 
donated exsiccata and collections, was kept separately and a taxonomic criterion 
was not applied to the Herbarium’s arrangement. Between 1910 and 1918 the 
collections were merged, and all specimens were arranged taxonomically (Kohler 
2001a). 

After the Second World War the fungal collection was separated from plant 
collections and arranged by Barbara Guminska, who in 1950 became the first 
curator of the Herbarium’s mycological section (Kohler 2001b) and served 


128 ... Ociepa, Zubek & Mleczko 


PLATE 1. The oldest specimen in the fungal collection of the Jagiellonian University Herbarium: 
“Geastrum hygrometricum Pers. Normandie. 1807. D. Delaroche (?). Habitat in Italia, Helvetia, 
Germania umbrosis. 4.” 


as curator until her retirement in 1993. The next curator, Katarzyna Turnau 
(1993-2005), was succeeded by Piotr Mleczko, who currently holds this post. 

Until 2006 the specimens in the mycological collection were not numbered. In 
that year all specimens were counted and most of them were assigned numbers. 
The collection now contains 20,195 numbered specimens: 5564 Ascomycota, 
11,098 Basidiomycota, 2682 anamorphic fungi, and 851 fungus-like organisms 
(mostly Peronospora in the Oomycota). In addition it holds also about 900 
unnumbered macromycete specimens collected by Barbara Guminska in Pieniny 
National Park, Poland. 


The collections 

One of the most valuable fungal collections is the Javanese collection 
of Marian Raciborski. Raciborski’s collection dates back to 1896-1900 and 
consists of dried specimens marked with ‘J’ (Java) after the number (Fic. 2) 
as well as some samples preserved in ethanol-formaldehyde solution, which 
are in the Botanical Garden Museum (Kohler & Zemanek 1989). According 
to Wojewoda (1986), Raciborski described 235 new fungal taxa from Java, all 
tropical microfungi except for one macromycete. Many genera and species 
described by Raciborski are still accepted under his names: e.g. Aldona 


KRA fungal collection (Poland) ... 129 


woe 


r 
i 
i 
7 
. 


= “Tolyposporvum bogorience Rac, 
Bull Al Acad, Sq) Cracow'e 41909:349, 
1404 


determ. Kalman Vanky 
A697] 192% 


olyposporium bogoriense Racib.— HOLOTYPUS! 
' = Sporisorium bogoriense (Racib.) M. Piatek 


i 
i Rev. Marcin Piatek : 
W. Szafer Institute of Botany PAS, Krakow 26.VII.2005 


PLATE 2. A specimen from the Javanese collection of Marian Raciborski: “Ustilago bogoriense Rac., 
Panicum sp., Bogor.’ 


(A. stella-nigra), Alina (A. jasmini), Anhellia (A. tristis), Balladyna (B. gardeniae), 
Farysia (F. javanica), Goplana (G. aporosae, G. micheliae, G. mirabilis), Lambro 
(L. insignis), Ordonia (O. orthobasidion), Skierka (S. agallocha, S. canarii), and 
Telimena (T: erythrinae) (Index Fungorum 2011). 

Other important exsiccata kept in KRA are those of Karl Wilhelm Gottlieb 
Leopold Fuckel, Otto Jaap, Gottlob Ludwig Rabenhorst, Heinrich Rehm, Pier 
Andrea Saccardo, Trajan Savulescu, Hans Sydow, and Charles Wright (see the 
list below), which comprise many syntypes. At present only the exsiccati of 
Ascomycota are catalogued, comprising 134 syntypes. 

The fungal section also holds many specimens gathered in Poland, including 
three holotypes: Exobasidium dubium Racib., Wawelia regia Namysl. (an exotic 
species found in the Botanical Garden of the Jagiellonian University, Krakow), 
and Geopyxis rehmii Turnau. The most important Polish collections were 


130 ... Ociepa, Zubek & Mleczko 


contributed by Wladystaw Wojewoda (1932-2010) from Ojcéw National Park 
(macromycetes), Katarzyna Turnau from the Gorce Mts. (larger ascomycetes), 
and Barbara Guminska from Pieniny National Park. Recently Piotr Mleczko 
and co-workers made a significant collection of fungi, mostly from southern 
Poland, which is now being incorporated into the Herbarium and will increase 
the number of specimens by approximately 1000. 

The most important fungal exsiccata and collections in the Herbarium 
mycological section are listed below. It should be noted that the Taxonomic 
Literature series (Stafleu & Cowan 1976-88; Stafleu & Mennega 1992-2000) and 
the monographs by Stevenson (1971) and Pfister (1985) mostly do not mention 
the sets stored at Jagiellonian University Herbarium. 

The names of fungal collections, dates, and authors follow (where possible) 
Stafleu & Cowan (1976-88), Stafleu & Mennega (1992-2000), Pfister (1985), or 
Stevenson (1971). Otherwise data are based on exsiccata labels or, with respect to 
author life spans, from various sources cited with each collection). 


Abbreviations 
IF - Index Fungorum (www.indexfungorum.org) 
SC-1-7 — Stafleu & Cowan (1976-88), vols. 1-7 
Pf — Pfister (1985) 
St — Stevenson (1971) 


POLISH COLLECTIONS 


* Fungi bialowiezenses exsiccati, Wincenty Siemaszko (1887-1943) [Kohler 2002, Pf, 
SC-5]. 

¢ Fungi parasitici Poloniae exsiccati, Marian Raciborski (1863-1917) [Piekietko- 
Zemanek 1986, Wojewoda 1986]. 

¢ Mycotheca polonica, Marian Raciborski and Boleslaw Namystowski (1882-1929) 
[Kohler 2002, Wojewoda 1986]. 

¢ Mycotheca polonica, Jézef Kochman (1903-95) and Boguslaw Salata (1940-99) 
[Majewski 1995, Chmiel & Mulenko 1999, Pf]. 


EUROPEAN COLLECTIONS (EXCEPT POLAND) 


* Ascomyceten, Ascomycetes exsiccatae, Heinrich Rehm (1828-1916) [St; Pf; SC-6]. 

* Cryptogamae Cechoslovenicae exiccatae, Frantisek Smarda (1904-68) [SC-5]. 

¢ Flora Hungarica, Gabriel Bohus (1914-2005) [IF]. 

¢ Flora hungarica exsiccata. Sectio botanica Musei Nationalis Hungarici, Budapest. 

¢ Flora Lituana Exsiccata Fungi parasitici, Antanas Minkevicius (1900-98) [Botanikos 
ir genetikos katedra 2011; Pf]. 

¢ Flora Romaniae exsiccata, Alexandru Borza (1887-1971) [Pop 1972; Pf; SC-1]. 

¢ Fungi bavarici exsiccati, Andreas Allescher (1828-1903) and Johann Nepomuk 
Schnabl (1853-99) [Pf; SC-1,5]. 

¢ Fungi bohemici, FrantiSek Bubak (1865-1925) [SC-1]. 


KRA fungal collection (Poland) ... 131 


¢ Fungi Europaei exsiccati Klotzschii herbarii vivi mycologici continuatio. Editio nova. 
Series secunda, Gottlob Ludwig Rabenhorst (1806-81) [Pf; St; SC-4]. 

¢ Fungi rhenani exsiccati, Karl Wilhelm Gottlieb Leopold Fuckel (1821-76) [SC-1]. 

¢ Fungi Schemnitziensis, Andreas Kmet (1841-1908) [Lizon 1973; Pf]. 

* Fungi selecti exsiccati, Otto Jaap (1864-1922), Carl Brick (1863-1924) [Pf; SC-2]. 

* Griby Rossii, Gavril Stepanowich Nevodovskij (1874-1952) and Voldemar Tranzschel 
(1868-1942) [Karczmarz & Salata 1982; Pf; SC-6]. 

¢ Herbarium mycologicum romanicum, Trajan Savulescu (1889-1963) [Pf SC-5]. 

Klotzschii herbarium vivum mycologicum, Gottlob Ludwig Rabenhorst [Pf St; 

SC-4]. 
Klotzschii herbarium vivum mycologicum, editio novo, Gottlob Ludwig Rabenhorst 
[Pf; St; SC-4]. 

° Kryptogamae exsiccatae editae a Museo Palatino Vindobonensi, Ginter von Beck 
(1856-1931), Alexander Zahlbruckner (1860-1938), Karl von Keissler (1872- 
1965), Franz Petrak (1886-1973) [Pf; St; SC-1,2,7]. 

¢ Mycotheca germanica, Paul Sydow (1851-1925), Hans Sydow (1879-1946) [Pf 
SC-6]. 

¢ Mycotheca veneta, Pier Andrea Saccardo (1845-1920) [Pf; SC-4]. 

Plantae cryptogamicae, quas in Arduenna collegit (Plantae cryptogamicae Arduennae), 

Marie-Anne Libert (1782-1865) [Pf; SC-3]. 


EXTRA-EUROPEAN COLLECTIONS 


* Cryptogamae parasiticae in Insula Javae lectae exsiccatae, Marian Raciborski [Pf] 
¢ Fungi cubenses wrightiani, Charles Wright (1811-86) [St; SC-7]. 

¢ Fungi exotici exsiccati, Hans Sydow [St; Pf; SC-6]. 

* Ohio Fungi, William Ashbrook Kellerman (1850-1908) [St; Pf; SC-2]. 


Loan requests should be directed to: Institute of Botany Herbarium (Fungal 
collection), Jagiellonian University, Kopernika 31, 31-501 Krakow, Poland. 
Curator of the mycological collection: Dr. Piotr Mleczko. 

The Herbarium web-page is http://www.ib.uj.edu.pl/en/?¢d=zielnik 


Acknowledgments 

Anna Maria Ociepa would like to thank Professor Adam Zajac for employment 
in Herbarium of Jagiellonian University in 2006. The authors wish to thank Professor 
Reinhard Agerer and Professor Reinhard Berndt for their very valuable comments on 
the manuscript. 


Literature cited 

Botanikos ir genetikos katedra. 2011. Profesorius Antanas Minkevicius [http://www.bg.gf. 
vu.lt/?Personalas:Anks%C4%8 Diau_dirb%C4%99_i%26scaron%3Bkiliausi_katedros_ 
%C5%BEmon%C4%97s:Profesorius_Antanas_Minkevi%C4%8Dius (viewed online on 4 
November 2011)]. (in Lithuanian) 

Chmiel MA, Mulenko W. 1999. Professor Boguslaw Salata (1940-1999). Acta Mycologica 34(1): 
3-5. 


132 ... Ociepa, Zubek & Mleczko 


Index Fungorum 2011. [http://www.indexfungorum.org/Names/Names.asp (viewed online 4 
November 2011)]. 

Karczmarz K, Satata B. 1982. Studies and botanical achievements of G.S. Nevodowski (1874-1952). 
Annales Universitatis Mariae Curie-Sklodowska, Sectio C, Biologia 37: 223-231. (in Russian, 
with English summary) 

Kohler P. 1999. History of the Jagiellonian University Herbarium (KRA) (Cracow, Poland) in 
1780-1910. Kwartalnik Historii Nauki i Techniki 44(2): 7-60. (in Polish with English 
summary) 

Kohler P. 2001a. History of the Jagiellonian University Herbarium (KRA) (Cracow, Poland) 
in 1910-2000. Kwartalnik Historii Nauki i Techniki 46(4): 77-104. (in Polish with English 
summary) 

Kohler P. 2001b. History of the Jagiellonian University Herbarium (KRA), Cracow, Poland. Taxon 
50: 943-945. 

Kohler P. 2002. Botany at the Academic Society of Cracow, Academy of Sciences and Letters and 
the Polish Academy of Sciences and Letters (1815-1952). Studia i materialy do dziejéw Polskiej 
Akademii Umiejetnosci. Tom II. Polska Akademia Umiejetnosci, Krakow. (in Polish with 
English summary) 

Kohler PS, Zemanek A. 1989. The Marian Raciborski collection in the Museum of the Jagiellonian 
University Botanical Garden. Zeszyty Naukowe UJ 926, Prace Botaniczne 18: 135-148. (in 
Polish with English summary) 

Lizon P. 1973. Andrej Kmet a mykologicky vyskum Slovenska. Ceska Mykologie 27(3): 177-179. 

Majewski T. 1995. Jézef Kochman (1903-1995). Rocznik Towarzystwa Naukowego Warszawskiego 
58: 55-57. (in Polish) 

Pfister DH. 1985. A bibliographic account of exsiccatae containing fungi. Mycotaxon 23: 1-139. 

Piekietko-Zemanek A. 1986. A calendar of life of Marian Raciborski. 9-43, in: J Kornas (ed.): 
Marian Raciborski. Studia nad zyciem i dzialalnoscia naukowa. Zeszyty Naukowe UJ, Varia 
210. (in Polish with English summary) 

Pop E. 1972. In memoriam Elexandre Borza (1887-1971). Vegetatio 25(5-6): 273-277. (in French) 
http://dx.doi.org/10.1007/BF02198407 

Stafleu FA, Cowan RS. 1976-88. Taxonomic literature. Volume I-VII. Regnum Vegetabile 94-116. 

Stafleu FA, Mennega EA. 1992-2000. Taxonomic literature. Supplement I-VI, Regnum Vegetabile 
125-137. 

Stevenson JA. 1971. An account of fungus exsiccati containing material from the Americas. Beihefte 
zur Nova Hedwigia 36: 1-563. 

Wojewoda W. 1986. Mycology in the scientific output of Marian Raciborski. 59-78, in: J Kornas 
(ed.): Marian Raciborski. Studia nad zyciem i dzialalnoscia naukowa. Zeszyty Naukowe UJ, 
Varia 210. (in Polish with English summary) 


ISSN (print) 0093-4666 © 2012. Mycotaxon, Ltd. ISSN (online) 2154-8889 


MY COTAXON 


http://dx.doi.org/10.5248/120.133 
Volume 120, pp. 133-137 April-June 2012 


Mycena pseudoinclinata, new to Italy 


ALFONSO LA Rosa, ALESSANDRO SAITTA, RICCARDO COMPAGNO 
& GIUSEPPE VENTURELLA* 


Dipartimento di Biologia vegetale e Biodiversita, Universita di Palermo, 
Via Archirafi 38, I-90123 Palermo, Italy 
* CORRESPONDENCE TO: giuseppe. venturella@unipa. it 


ABSTRACT - The first report of Mycena pseudoinclinata in Italy is accompanied by notes on 
the taxonomy, ecology, and distribution of this uncommon basidiomycete. 


Key worps — Mycenaceae, sect. Fragilipedes, basidiomycetes 


Introduction 

Mycena pseudoinclinata (Mycenaceae Overeem) belongs to sect. Fragilipedes 
(Fr.) Quél., which is considered the largest section in Mycena (Pers.) Roussel. 
The species was originally described by Smith (1947), who distinguished it from 
M. inclinata (Fr.) Quél. by the presence of both pleurocystidia and cheilocystidia 
and absence of (or fewer) fibrillose flecks on the stipe. This taxon has a limited 
distribution all over the world, particularly in Europe where it has recently been 
recorded only in Switzerland and France (Robich 2003). This paper deals with 
the first record of M. pseudoinclinata in Italy, which we recently collected in a 
reforested area in the neighborhood of the town of Palermo (Sicily). 


Materials & methods 

Basidiomata were identified while fresh and microscopic features were observed 
in H,O using a Leica DMLB microscope; spore measurements were based on 
100 observations. Nomenclature follows the Index Fungorum (http://www. 
indexfungorum.org/Names/Names.asp). The description of the characteristics of 
the species is based on personal observations that were subsequently compared 
with the descriptions given by Smith (1947) and Robich (2003). The collection is 
stored in the fungal dried reference collection of the Herbarium Mediterraneum 
Panormitanum, Palermo (PAL). 


134 ... La Rosa & al. 


Taxonomy 


Mycena pseudoinclinata A.H. Sm., North Amer. Species Mycena: 327. 1947. 
Fics. 1B, 2 

Pileus 20-30 mm, ash-gray, conical to campanulate, pruinose, surface moist 
and glabrous, disc slightly umbonate, margin striate. Lamellae close, white, 
notched, margin concolorous to the face of lamellae; lamellulae present. Stipe 70 
x 3 mm, fragile, hollow, almost concolorous with the pileus, darker at the base 
with a slight whitish felt. Flesh typically with a farinaceous odor and taste. 

Basidiospores 8-10 x 6-6.5 um, ellipsoid to broadly ellipsoid, smooth, 
amyloid. Basidia, 30-35 x 7.5-8.5 um, tetrasporic, claviform. Cheilocystidia 
20-60 x 9-12 um, claviform, sphaerical-pedicellate, with irregular excrescences 
of different shapes < 10-15 um in length, well-spaced. Epicutis composed of 
cylindrical hyphae, smooth, 2-5 um diam., with terminal elements 50-70 x 
2-6 um. Subhymenial layer with sub-globose hyphae, isodiametric, 3-8 um 
diam. Hyphae of stipe cylindrical, innermost 8-25 um and outermost 1.5-2.5 
um diam., smooth or with sparse excrescences, dextrinoid. 

COLLECTION EXAMINED: ITALY. Sicity: Raffo Rosso, neighborhood in Palermo, 423 

m, on rotten trunks of Eucalyptus camaldulensis Dehnh. (Myrtaceae), 13 Jan 2011, GPS: 

38°10'46"N 13°15'40"E, coll. R. Compagno & A. La Rosa (PAL 960). 
ComMENTs — Robich (2003) included mycenas with large basidiomata in sect. 
Mycena. Some species that might be confused with others in sect. Filipedes 
(Fr.) Quél. or Fragilipedes can be differentiated by their basidioma shape and 
cheilocystidial type. Mycenas in sect. Fragilipedes are primarily differentiated 
through cheilocystidial and/or pleurocystidial shapes, here the main character 
differentiating M. pseudoinclinata is the numerous large irregular protuberances 
on the cheilocystidia. Although Smith (1947) referred to pleurocystidia in his 
original description of M. pseudoinclinata, Maas Geesteranus (1988a,b) correctly 
described this species as lacking them. Robich, who cited the cheilocystidia as 
restricted to the lamellar edge (Robich 2003), includes the species in his revised 
key to Mycena sect. Fragilipedes of the northern hemisphere. 

Smith (1947) first reported M. pseudoinclinata as densely cespitose and 
occurring in Tennessee, Pennsylvania, and Michigan (U.S.A.). Bi et al. (1993) 
report the species from China on fallen twigs in broad-leaved woods. In Europe 
Gerault (2005) reports the species from France and Switzerland in broad-leaved 
forests mainly characterized by oaks. 

We found that M. pseudoinclinata also grows in Eucalyptus plantations 
(Fic. 1A) and confirm its role in decaying wood and its typical cespitose habit, 
as demonstrated by our collection of eight connate basidiomata per trunk 
(Fic. 1B). The literature notes that M. pseudoinclinata fruits from spring (April) 
through autumn; in Sicily we have collected basidiomata as early as January, no 


Mycena pseudoinclinata, new to Italy ... 135 


Fic. 1: Mycena pseudoinclinata. 
A. Habitat in Eucalyptus camaldulensis plantation. B. Basidiomata. 


136 ... La Rosa & al. 


10 um 


O® 
ay 
“8 


d -—— 10m 


Fic. 2: Mycena pseudoinclinata (PAL 960(Mic)]. 
a. Basidiospores. b. Claviform cheilocystidia with irregular excrescences of different shapes. 
c. Epicutis with cylindrical hyphae. d. Vesiculose and swollen hyphae in the pileitrama. 


Mycena pseudoinclinata, new to Italy ... 137 


doubt due to the mild weather conditions on the island. Our report represents 
the southernmost limit of its distribution in Europe. 


Acknowledgements 

The authors wish to thank Dr Vladimir Antonin (Czech Republic) and Dr Stephanos 
Diamandis (Greece) for critically reviewing the manuscript. The authors would also like 
to thank Dr. Cassandra Funsten (USA) for her help with linguistic revision. 


Literature cited 

Bi ZS, Zheng GY, Li TH. 1993. The macrofungus flora of China's Guangdong Province. The Chinese 
University Press, Shatin, N.T., Hong Kong. 737 p. 

Gerault A. 2005. Florule evolutive des Basidiomycotina du Finistére. Heterobasidiomycetes. 
Tricholomatales. Vers. 2.1. 205 p. http://projet.aulnaies.free.fr/Florules/TRICHOLOMATALES. 
pdf 

Maas Geesteranus RA. 1988a. Conspectus of the mycenas of the northern hemisphere - 9. Section 
Fragilipedes, species A-G. Proc. Kon. Ned. Akad. Wetensch., Ser. C, 91: 43-83. 

Maas Geesteranus RA. 1988b. Conspectus of the mycenas of the northern hemisphere - 9. Section 
Fragilipedes, species I-R. Proc. Kon. Ned. Akad. Wetensch., Ser. C, 91: 129-159. 

Robich G. 2003. Mycena d’Europa. Associazione Micologica Bresadola, Trento. 728 p. 

Robich G. 2006. A revised key to the species of Mycena, section Fragilipedes of Northern hemisphere. 
Persoonia 19: 143. 

Smith AH. 1947. North American species of Mycena. Ann. Arbor: University of Michigan Press. 
London: Geoffrey Cumberlege, Oxford University Press. 521 p. 


ISSN (print) 0093-4666 © 2012. Mycotaxon, Ltd. ISSN (online) 2154-8889 


MY COTAXON 


http://dx.doi.org/10.5248/120.139 
Volume 120, pp. 139-147 April-June 2012 


Rossbeevera yunnanensis (Boletaceae, Boletales), 
a new sequestrate species from southern China 


TAKAMICHI ORIHARA*”?, MATTHEW E. SMITH3, 
ZAI-WEI GE* & NITARO MAEKAWA? 


"Kanagawa Prefectural Museum of Natural History, 
499 Iriuda, Odawara-shi, Kanagawa 250-0031, Japan 
*The United Graduate School of Agricultural Sciences, Tottori University, 
4-101 Koyama-cho-minami, Tottori 680-8553, Japan 
*Department of Plant Pathology, University of Florida, Gainesville FL 32611-0680, USA 
‘Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China 
°Fungus/Mushroom Resource and Research Center, Faculty of Agriculture, Tottori University, 
4-101 Koyama-cho-minami, Tottori 680-8553, Japan 
* CORRESPONDENCE TO: t_orihara@nh.kanagawa-museum.jp 


ABSTRACT — A new sequestrate (truffle-like) species, Rossbeevera yunnanensis, is described 
based on a collection from Chuxiong Prefecture, Yunnan Province, China. The species is 
morphologically characterized by its large, narrow, fusoid to fusiform basidiospores and 
remarkably thin peridium. Maximum likelihood and neighbor joining phylogenies of our 
nLSU rDNA dataset indicate that the species constitutes the earliest diverging lineage within 
the genus Rossbeevera and has a close phylogenetic relationship to species of Leccinellum. 
Morphological and phylogenetic relationships to the other Asian members of Rossbeevera 
are discussed. 


Key worps — Chamonixia, FTA, hypogeous fungi, Leccinum, Rosbeeva 


Introduction 

The sequestrate (truffle-like) genus Rossbeevera T. Lebel & Orihara (erroneous 
orthographic variant: “Rosbeeva”) was erected to accommodate Australasian 
and Asian species of Chamonixia Rolland (Lebel et al. 2012a,b), which are 
phylogenetically close to but distinct from Chamonixia sensu stricto within 
Boletaceae. The genus is characterized by ellipsoid to fusiform basidiospores 
with 3-5 longitudinal ridges, bluish green to cyanescent discoloration of 
basidiomata, and a thin cutis-like whitish peridium. Phylogenetically, this genus 
is closely related to the epigeous bolete genera Leccinum Gray and Leccinellum 
Bresinsky & Manfr. Binder and sequestrate genera Chamonixia and Octaviania 
Vittad. (Lebel et al. 2012a, Orihara et al. 2012). Rossbeevera species have 


140 ... Orihara & al. 


been previously reported from Australia—R. vittatispora (G.W. Beaton et al.) 
T. Lebel and R. westraliensis T. Lebel, New Zealand—R. pachydermis (Zeller 
& C.W. Dodge) T. Lebel, Southeast Asia—R. mucosa (Petri) T. Lebel, China— 
R. bispora (B.C. Zhang & Y.N. Yu) T. Lebel & Orihara, and Japan—R. eucyanea 
Orihara and R. griseovelutina Orihara (Lebel et al. 2012a,b). 

During a fieldtrip in Yunnan Province, China, two of the authors (Smith & Ge) 
collected Rossbeevera basidiomata that could not be ascribed to any currently 
known species. Here we propose this material as a new species, Rossbeevera 
yunnanensis, based on morphological evidence as well as phylogenetic analyses 
of the large subunit of the nuclear ribosomal DNA (nLSU). 


Materials & methods 


Taxon sampling and macro- and microscopic characterization 

Fresh basidiomata were located and collected using a truffle rake. Specimens were 
placed in wax paper bags and transported to the laboratory within six hours where they 
were photographed and rapidly dried in a closed container with silica drying beads. 
The dried specimens are deposited in the Herbarium of Cryptogams, Kunming Institute 
of Botany, Chinese Academy of Sciences (KUN, with HKAS accession numbers), 
Kanagawa Prefectural Museum of Natural History (KPM), and the University of Florida 
Herbarium at the Florida Museum of Natural History (FLAS). 

For standard light microscopy and differential interference contrast microscopy, 
hand-cut sections of dried specimens were mounted in water, 3% KOH, lacto-glycerol, 
or 1% phloxine B aqueous solution. To determine the amyloid reaction, dried material 
was stained with Melzer’s reagent. Spore dimensions (length x width range) and their 
standard deviations (SD), and length of hilar appendages were measured based on 50 
randomly selected spores. The length : width ratio (Q) and hilar appendage: spore length 
ratio (HA/S, newly introduced here) are presented to describe a range of the basidiospore 
proportion. Measurements include the hilar appendage but not ornamentation or 
pedicel. Basidium ranges are given as length range x width. 


Molecular methods 

DNA was extracted from a dried basidioma using the Indicating FTA Cards 
(Whatman International Ltd, Maidstone, England). Internal tissue ca. 1 mm thick was 
soaked in 99.5% ethanol for 2 min before DNA extraction. The extraction was done 
according to the manufacturer's protocol for plant samples (available at http://www. 
whatman.com/References/WGI_1397_PlantPoster_V6.pdf) using FTA Cards, more 
convenient and efficient over traditional methods (Dentinger et al. 2010). Furthermore, 
we demonstrate here that this technique is useful for dried tissues after a brief ethanol 
pretreatment. For PCR amplification and sequencing reactions, we used the primer 
pair LROR / LRS (Vilgalys & Hester 1990). For PCR conditions, sequencing and editing 
of the obtained sequence, see Lebel et al. (2012a). The nLSU sequence is deposited in 
GenBank. 

Sequences included in the nLSU dataset were retrieved from GenBank based on the 
previous analyses of Rossbeevera and allied genera (Lebel et al. 2012a). Some sequences 


Rossbeevera yunnanensis sp. nov. (China) ... 141 


of Leccinellum spp., which are shown to be paraphyletic with Rossbeevera, were selected 
according to the phylogeny in Lebel et al. (2012a) and were used to root the phylogenetic 
tree. Multiple sequence alignment was performed using Clustal X ver. 1.83 (Thompson 
et al. 1997) and the data were manually adjusted in SeaView (Galtier et al. 1996). Gaps 
were treated as “missing” data for all analyses. 

The neighbor-joining (NJ) analysis based on p distance was conducted with Clustal 
X ver. 1.83 with 1000 bootstrap replicates and with default settings. Subsequently, the 
maximum likelihood (ML) analysis was done by PhyML ver. 3.0 (Guindon et al. 2010) 
under GTR + I + G model based on hLRT estimated by MrModeltest 2.3 (Nylander 
2004), starting the analysis using a tree estimated by the Nearest Neighbor Interchanges 
(NNIs) and setting the number of bootstrap replicates to 1000. The resulting trees were 
visualized with FigTree ver. 1.3.1 (Rambaut, available at http://tree.bio.ed.ac.uk/). 


Results 

The finally aligned nLSU dataset was 837 bp long, including full length 
reads for all 19 sequences but excluding the primer regions. Settings of the best 
model estimated by hLRT in MrModeltest 2.3 were as follows: base frequencies 
(A = 0.2700, C = 0.1941, G = 0.2904, T = 0.2455), proportion of invariable 
sites (I = 0.7687), and gamma distribution shape parameter (a = 1.9606). 
Maximum likelihood analysis generated one ML tree (In L = -2078.576887; 
Fic. 1). Branches that were moderately to strongly supported by ML bootstrap 
values (>60%) were also strongly supported in the NJ topology (BS >70%). In 
the Rossbeevera clade, R. eucyanea and R. griseovelutina were each supported to 
be monophyletic by high BS values but the Australasian species were not well 
differentiated from each other, as shown in Lebel et al. (2012a). The Chinese 
Rossbeevera sp. MES420 formed a basal branch within the Rossbeevera clade 
and was phylogenetically distinct from the other members of Rossbeevera 
included in this study. 


Taxonomy 


Rossbeevera yunnanensis Orihara & M.E. Sm., sp. nov. Fic. 2 
MycoBank MB 563649 
Differs from Rossbeevera bispora by thinner or partially lacking peridium, fusoid to 
fusiform basidiospores, and 2—4-spored basidia. 


Type: China, Yunnan Province, ChuXiong Yi Autonomous Prefecture, Mt. Zixi, 19 
Sept. 2010, M. E. Smith et Z.-W. Ge, MES420 (holotype - HKAS 70689, isotype - KPM- 
NC0017850, GenBank JN979437). 


ETyMOLOGy: yunnanensis (Latin) refers to the type locality. 


BASIDIOMATA Sparse, ca. 1 cm, subglobose, soft, surface covered with very thin, 
minutely felty, whitish to almost translucent peridium gradually turning bluish 
gray or black when touched or bruised, the peridium partially absent, exposing 
the gleba. GLEBA off-white to beige when immature, reddish brown to blackish 


142 ... Orihara & al. 


Rossbeevera westraliensis AUS HQ647161 


Rossbeevera westraliensis AUS HQ647160 
Rossbeevera vittatispora AUS HQ647148 
Rossbeevera pachydermis NZ DQ534620 


53/ - 


pate Rossbeevera pachydermis NZ HQ647157 


Rossbeevera vittatispora AUS HQ647150 
Rossbeevera vittatispora AUS HQ647159 
Rossbeevera griseovelutina JPN HQ693877 
100/100 | Rossbeevera griseovelutina JPN HQ693876 


Rossbeevera griseovelutina JPN HQ693878 
96/97 


Rossbeevera eucyanea JPN HQ693880 
100/100 ' Rossbeevera eucyanea JPN HQ693879 


Rossbeevera yunnanensis CHINA JN979437 


Leccinellum albellum AY612811 


Leccinellum aff. griseum JPN JN378509 
Leccinellum carpini AF139691 


Leccinellum rugosiceps AY612813 


Leccinellum crocipodium AF139694 


a oat ee 100/100 L Leccinellum crocipodium AF454590 
0.1 substitutions / site 


FiguRE 1. Maximum likelihood (ML) tree of the nLSU dataset of Rossbeevera species with 
Leccinellum species selected as the outgroup. ML and Neighbor-Joining (NJ) bootstrap (BS) values 
(1000 replicates; only BS >50% are shown) are indicated above or below branches or at nodes as 
MLBS/NJBS. Abbreviations: AUS = Australia; NZ = New Zealand; JPN = Japan. 


brown at maturity, rubbery, composed of minute, irregular locules, turning 
bluish gray in some portions when cut and exposed to air, trama subgelatinous, 
somewhat translucent. STIPE-COLUMELLA present but reduced, somewhat 
dendroid or as a small basal pad, subgelatinous, translucent. ODOR unknown. 
BASIDIOSPORES (14.4—)16.9-23.4(-24) x 6.8-9(-9.5) um, (mean + SD 
= 20.1 + 1.60 x 7.8 + 0.61), Q = 2-3.8 (mean = 3.1), symmetric, fusoid to 
fusiform, inamyloid, nondextrinoid, colorless at first then becoming reddish 
brown at maturity, with 3-4 or rarely 5 smooth, longitudinal ridges up to 2.3 
um high (mostly ca. 1 um high) in water, walls 0.7-1.5 um thick, with a large 
hilar appendage (2-)2.3-3.8 um long (mean 3.1), HA/S = 0.12-0.22, (mean = 
0.16) at the base and a minute, incomplete hollow (> 1 um in diameter) at the 
tip. Basrpra 17.5-30 x 6.8-11 um (mean = 23.5 x 8.4 um; n = 15), cylindrical 
to cylindro-clavate, colorless to pale yellow-brown to reddish brown, 2-, 3- or 


Rossbeevera yunnanensis sp. nov. (China) ... 143 


4-spored. HymEenium developed when immature but collapsed at maturity, 
colorless, hymenial cystidia not seen; BASIDIOLES cylindrical or clavate to 
clavulate. SUBHYMENIUM not developed. TRama of densely or somewhat 
loosely interwoven, partly branched, colorless, non-inflated, thin-walled (< 0.8 
uum thick), filamentous hyphae 3.5-8 um broad. STIPE-COLUMELLA composed 
of loosely interwoven, subgelatinous, thin-walled (< ca. 1 um thick) filamentous 
hyphae (2-)5-8(-9.5) um broad. PERIDIUM thin, absent in some portions, up 
to 60 um thick in the dried basidioma, yellowish brown under light microscopy, 
composed of repent, non-inflated, thin-walled (< 0.8 um thick) filamentous 
hyphae 2.5-6 um broad but the hyphae are collapsed in most portions. CLAMP 
CONNECTIONS absent in all tissues. 

HABITAT AND DISTRIBUTION: Hypogeous in mixed forest dominated by 
Pinus yunnanensis Franch., Lithocarpus mairei (Schottky) Rehder, L. dealbatus 
(Hook. f. & Thomson ex Miq.) Rehder, and unidentified Quercus spp.; known 
only from Yunnan Province, China. 


Discussion 

Rossbeevera yunnanensis shares several morphological characteristics 
with the other Rossbeevera species, including the bluish color change of the 
peridium and reddish brown basidiospores with 3-5 unbranched longitudinal 
ridges. ‘This new species also has the large, distinctly fusoid to fusiform (Q = 
2.0-3.8) basidiospores and remarkably thin peridium characteristic of the 
genus. Rossbeevera griseovelutina, which is reported from Japan, has slightly 
larger basidiospores (14.4-31.9 x 6.7-10.4 um, mean = 22.2 x 8.7 um) and the 
Q value is smaller (Q = 2.0-3.3; Lebel et al. 2012a). In addition, the latter species 
has a more fully developed peridiopellis composed of more or less vertically 
oriented, partially inflated hyphae. Rossbeevera yunnanensis also differs 
morphologically from another known Chinese species, R. bispora, because 
R. yunnanensis has a thinner peridium, larger and narrower basidiospores, and 
its basidia often bear 4 spores rather than 2 spores characteristic of R. bispora 
(Zhang & Yu 1989). We have examined the holotype of R. bispora (GDGM5688) 
and have confirmed the above-noted differences. The Southeast Asian species, 
R. mucosa is readily distinguished from R. yunnanensis in its ellipsoid to fusoid 
basidiospores (13—)15-17 um in diameter with a lower Q value (Q = 1.76-2.05; 
Lebel et al. 2012a). 

The HA/S ratio, which is newly introduced in this study, could be helpful to 
describe spore shapes objectively when spore sizes vary widely within collections 
or sporocarps of a particular species. Basidiospores of R. griseovelutina are 
somewhat similar to those of R. yunnanensis as discussed above, but the 
mean HA/S ratio of R. griseovelutina was significantly higher than that of 
R. yunnanensis. However, the ranges overlapped: HA/S of R. griseovelutina = 
0.12-0.24, mean 0.18, n = 25 vs. HA/S of R. yunnanensis = 0.12-0.22, mean 


144 ... Orihara & al. 


= 0.16. Basidiospore morphology is widely used as a key character for the 
taxonomy of sequestrate fungi (Castellano et al. 1989). The HA/S difference 
is proposed as a new, useful diagnostic character that could be useful in the 
taxonomy of Rossbeevera and other sequestrate genera. 

In our phylogeny R. yunnanensis formed the earliest diverging lineage 
within the Rossbeevera clade with moderate to strong bootstrap support 
(ML: 67%; NJ: 91%). Although we have not been able to include sequences of 
R. bispora, it is possible that the two Chinese species are closely related within 
Rossbeevera, because they share a similar overall morphology, including a cutis- 
like peridium and relatively large basidiospores with mostly 3-4 longitudinal 
ridges. Sequences from additional genes and specimens of Rossbeevera from 
China and the adjacent regions would help to clarify the evolutionary origin of 
this sequestrate genus. 

Although we have examined only one R. yunnanensis collection, its 
morphological characteristics and unique phylogenetic position are sufficient 
to discriminate the species from the other members of Rossbeevera. We would 
prefer to have additional specimens in order to provide more ecological 
information about R. yunnanensis, such as details of its phenology and host tree 
preferences. However, since hypogeous fungi are difficult to find and collect, we 
felt it was important to describe this new Rossbeevera species to promote further 
research on the systematics of Asian sequestrate fungi. Unique sequestrate 
fungi have been found in Asia for more than a century (e.g. Corner & Hawker 
1953; Smith & Schmull 2011). However, several recent studies highlight the fact 
that Asia is likely a hotspot for unique, endemic truffles and probably hosts a 
high diversity of undescribed sequestrate fungi (Yang et al. 2006; Desjardin et 
al. 2008, 2009; Orihara et al. 2008, 2010, 2012; Kinoshita et al. 2011). Clearly, 
more taxonomic work on sequestrate fungi is needed in this region. 


Acknowledgments 

The study was supported by Research Fellowships for Young Scientists (No. 21-6052) 
from the Japan Society for the Promotion of Science and partially by the Grant-in-Aid 
for the Global COE Program “Advanced Utilization of Fungus/Mushroom Resources 
for Sustainable Society in Harmony with Nature” from the Ministry of Education, 
Culture, Sports, Science and Technology of Japan. Financial support for the field trip 


FIGURE 2 (right). Rossbeevera yunnanensis (holotype): a. Basidiomata. A reduced columella is 
present in the center of the section (arrow); b. Significantly reduced peridium of dried material 
(arrow); c. Thick portion of the peridium; d. Mature basidiospores with longitudinal ridges 
(arrows) mounted in lacto-glycerol; e. Immature basidiospores viewed from above (arrow); 
f. 3- and 4-spored basidia mounted in lacto-glycerol after a pretreatment with 1% phloxine B 
aqueous solution; g. 2-spored basidium mounted in lacto-glycerol. Scale bars: a = 1 cm; b = 20 um; 
c = 30 um; d-g = 10 um. 


Rossbeevera yunnanensis sp. nov. (China) ... 145 


146 ... Orihara & al. 


was provided by the National Natural Science Foundation of China (No. 30800004) 
and the Knowledge Innovation Program of the Chinese Academy of Sciences (No. 
KSCX2-EW-J-24). The authors gratefully acknowledge Dr. Kentaro Hosaka (National 
Museum of Nature and Science, Ibaraki, Japan) and Ms. Rosanne Healy (University of 
Minnesota) for serving as pre-submission reviewers and their helpful comments on the 
manuscript. 


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ISSN (print) 0093-4666 © 2012. Mycotaxon, Ltd. ISSN (online) 2154-8889 


MY COTAXON 


http://dx.doi.org/10.5248/120.149 
Volume 120, pp. 149-155 April-June 2012 


Octaviania violascens: a new sequestrate bolete from Thailand 


RATTAKET CHOEYKLIN™, THITIYA BOONPRATUANG?’, 
SUJINDA SOMMAI’ & SAYANH SOMRITHIPOL’ 


‘Enzyme Technology and Waste Management Research Unit, 
Kasetsart Agricultural and Agro-Industrial Product Improvement Institute (KAPI), 
Kasetsart University, Bangkok, Thailand 
*Mycology Laboratory, Bioresources Technology Unit, 
National Center for Genetic Engineering and Biotechnology, 
Thailand Science Park, Pathumthani, Thailand 


* CORRESPONDENCE TO: rattaketster@gmail.com 


ABSTRACT — Octaviania violascens sp. nov. is proposed for a new sequestrate bolete 
from Khao Yai National Park, Nakhon Ratchasima Province, Thailand. A comprehensive 
description and illustrations are provided. 


Key worps — Basidiomycota, truffle, Octavianiaceae 


Introduction 

While walking along an elephant trail collecting mushrooms in a bamboo 
grove in Khao Yai National Park, Nakhon Ratchasima Province, ‘Thailand, 
we encountered a rose-colored fungus where recent rain had washed away 
the topsoil. The distinctive characteristics of this species, besides the rose 
color of its basidiocarps, were that it bruised deep reddish violet, contained 
non-glutinous gleba, and had clamp connections in the peridial layer and 
basidia containing only two highly ornamented spores. Its micromorphology 
clearly placed it in Octaviania and it is the first record of this genus from 
Thailand. Octaviania is included in the group of sequestrate basidiomycetes 
called truffles. In Thailand 8 species of sequestrate fungi have been reported, 
including Arcangeliella rosea (Harkn.) Zeller & C.W. Dodge (Ellingsen 1982), 
A. beccarii (Petri) Zeller & C.W. Dodge (Chandrasrikul et al. 2008), Descomyces 
cf. albellus (Massee & Rodway) Bougher & Castellano (Dissing 1963, Ellingsen 
1982), Mycoamaranthus cambodgensis (Pat.) Trappe, et al. (Chandrasrikul et al. 
2007, 2008, Lumyong et al. 2003), Radiigera tropica Orihara & T. Kasuya, and 
three other unknown species from Dissing (1963) in the genera Hymenogaster, 


150 ... Choeyklin & al. 


Leucogaster, and Melanogaster. This paper describes a newly discovered species, 
Octaviania violascens, from Thailand. 


Materials & methods 

COLLECTION SITE: The study site is in a bamboo grove, in a secondary forest near 
the 28 km marker post along the road from the northern entrance in Khao Yai National 
Park, Nakhon Ratchasima Province, Thailand. 

FUNGAL MATERIALS: Dried specimens are deposited in the BIOTEC Bangkok 
Herbarium (BBH). 

MorpHo ocy: Macromorphological data were recorded from dried specimens using 
an Olympus model XZ30 stereomicroscope. Micromorphological data were recorded 
from dried specimens using an Olympus model BX51 microscope. Drawings were 
made using an Olympus Camera Lucida model U-DA. Measurements and drawings 
were made from slide preparations stained with cotton blue, Melzer’s reagent, or 3% 
potassium hydroxide. The following abbreviations are used for chemical reactions: IKI 
(Melzer’s reagent, with IKI- = non—amyloid and non-dextrinoid; IKI+ = amyloid or 
dextrinoid), KOH (3% potassium hydroxide), and CB (cotton blue; CB+ = cyanophilous; 
CB(+) = weakly cyanophilous; CB- = acyanophilous). Basidium width was measured 
at the broadest part, and basidium length was measured from the apex (sterigmata 
excluded) to basal septum. Color terms and notations in parentheses follow Kornerup 
and Wanscher (1963). Spore statistics include: x = the arithmetic mean of the spore 
length by spore width (+ standard deviation) for n spores measured; Q = the quotient of 
length and width in any one spore, indicated as a range of variation in n spores measured; 
Qm = the mean of Q—values (+ standard deviation). Ultrastructures were recorded from 
dried specimens using a Hitachi S-3400N scanning electron microscope. 


Taxonomy 


Octaviania violascens Choeyklin, Boonprat. & Somrith., sp. nov. PLATES 1-3 
MycoBank MB 563238 
Differt a O. purpurea, absque columella et rhizomorpha, loculus rotundatus, spora 


verruca angulari, basidia bispora pariete crasso; praesentia fibularum et cellularum 
spheropedunculatarum et hypharum laticiferarum. 


TYPE — THAILAND. Nakhon Ratchasima, Pak Chong district, Khao Yai National Park, 28 
km. marker post (Bambusa spp. forest), 14°30'59.47"N 101°22'08.29"E, 23 August 2010, 
coll. Rattaket Choeyklin & Sujinda Sommai, RCK 00093 (Holotype BBH 30342). 


EtryMoLocy -— violascens (Latin) = turning violet. 


Basidiocarps 9-18 mm tall x 12-22 mm wide (dried specimens), ovoid, 
subglobose to turbinate, white to cream colored near base, lumpy or folded on 
the upper part; smooth to cracking to form squamules, the squamules reddish 
grey to greyish rose (12B2-3), staining reddish violet to deep violet (18C-D8) 
where bruised or cut; dull, dry, hard and cartilaginous. Peridium thin, 0.5-0.6 
mm diam., white, quickly changing from violet white to pale violet (16A3-4) 
when cut. Gleba lacunate, 0.5-1 mm diam., locules filled with powdery brown 


Octaviania violascens sp. nov. (Thailand) ... 151 


PLaTE 1: Octaviania violascens. 1. Basidiocarps in various shapes; 2. Peridial layer cracking to 
form squamules and deep violet stains where bruised; 3. Gleba lacunate, quickly changing to violet 
brown when cut, basidiocarps without rhizoid (holotype). Bar = 10 mm. 


spores when dried, dark brown when fresh, solid when fresh and with a hollow 
central cavity when dried, watery, quickly changing to violet brown (11F8) when 
cut; sterile tissue between locules fibrous, white but immediately changing to 
reddish violet to deep violet when bruised (18C-D8). Latex absent. Columella 
absent. Odor not distinctive. 

Basidiospores without spines (11-)12-15(-19) x 11-14(-15) um (x = 13.84 
+ 1.53 x 12.44 + 1.16 um, Q = 1.0-1.46, Qm = 1.09 + 0.11, n = 25); with spines 
(12-)15-17(-18) x (10-)15-20 um (x = 15.79 + 1.36 x 16.40 + 2.14 pm, Q = 
0.67-1.50, Qm = 0.98 + 0.15), orthotropic and heterotropic, symmetric and 
asymmetric, globose to subglobose, walls 2.0-2.5 um thick, pale yellowish 
brown, golden-brown, brown to dark brown in water and 3% KOH, IKI+ 
(weakly dextrinoid), CB-, spore ornamentation echinulate to conical spines, 
1-2 um tall x 2 um wide at base x 1 um wide at apex, most apical spines acute 


152 ... Choeyklin & al. 


PLATE 2: Octaviania violascens (holotype). 1. Basidiospore formation; 2. 2-spored basidia; 3. Basidia 
shapes; 4. Spheropedunculate cell from peridial context; 5. Hymenium with basidia; 6. Oleiferous 
hyphae; 7. Skeletal hyphae. Bar = 10 um. 


but some blunted or slightly bent, hyaline in 3% KOH and water, IKI+ (weakly 
dextrinoid), CB+; pedicel 6-10 x 3 um, walls 1-1.5 um thick, pale yellowish- 
brown. Basidia (12.5-)20-31(-35) x (5-)7.5-15 um, 2-spored, lageniform 
to fusoid, walls >1 tm thick, hyaline when young to pale yellowish brown to 
brown when mature in water and KOH, IKI-, CB+, basally septate; sterigmata 
7-8 um tall x 3 um wide, walls 1 um thick, pale brown in water and 3% KOH, 
IKI-, CB+, septa present at the sterigmal base. Peridial context 170-300 um 
diam., containing two types of hyphae consisting of oleiferous and binding 
hyphae; oleiferous hyphae 3-4 um wide, thin-walled, brown in water and 3% 


Octaviania violascens sp. nov. (Thailand) ... 153 


PLATE 3: Octaviania violascens (holotype): scanning electron micrographs of spores. 1. Mature 
basidiospore, 2. Basidiospore wall, 3. Basidiospores variation, Bar 1-2 = 10 um, 3 = 3 um 


KOH, IKI-, CB+; binding hyphae 4-6 um width, thick-walled, walls 1-1.5 um 
thick, hyaline in water and 3% KOH, IKI-, CB+, and pinkish when stained 
with phloxine. Sphaeropedunculate cells 27.5-30 x 27.5-30 um, embedded 
in the peridial context, pedicel 7 x 2 um, ovoid, thick-walled, hyaline, IKI-. 
Subhymenium IKI+ (weakly dextrinoid). Tramal plate 25-30 um diam., thin- 
walled, hyaline, IKI-. Peridiopellis of repent hyphae, interwoven, thin-walled, 
hyaline, IKI-. Sterile tissues that separate the locules are identical to the tramal 
plate. Clamp connections present only in the peridial tissue. 


154 ... Choeyklin & al. 


ECOLOGY & DISTRIBUTION: Solitary to gregarious or in small clusters, 
subhypogeous to hypogeous, in sandy soil in a Bambusa spp. (Bambusaceae) 
forest, in open secondary forest. Thailand, Nakhon Ratchasima Province, Khao 
Yai National Park, 28 km marker post. 


Discussion 

Octaviania violascens may be diagnosed by its echinulate spores with conical 
spines and thick-walled 2-spored basidia. The new species is most similar to 
O. purpurea Coker & Couch (Coker & Couch 1928) but differs in the texture 
of the gleba, absence of a columella and rhizomorphs, spore shape, colour, 
and ornamentation, presence of laticiferous hyphae, and basidia shape and 
sterigmata number (TaBLE 1). Spheropedunculate cells in the peridial context 
and clamp connections found in O. violascens are absent in O. purpurea 
(TABLE 1). 

Heliogaster Orihara & K. Iwase also produces an empty-chambered 
nonglutinous gleba, but unlike Octaviania its gleba is soft, not rubbery and its 
peridium composed of filamentous hyphae (not partly inflated hyphae with 
isodiametric cells) and spores with a perisporium. In the absence of DNA 
analyses, we retain O. violascens within Octaviania based on morphology. 


TABLE 1. Morphological characters in Octaviania violascens and O. purpurea. 


MORPHOLOGY O. violascens O. purpurea 
Sporocarp texture Rubbery Not reported 

Spore ornamentation Echinulate with conical spines Angular warts 

Spore shape Globose Subglobose 

Spore color Pale yellowish brown to brown Yellowish brown to dark brown 
Basidia 2-spored, thick-walled 1-spored, thin-walled 
Basidial shape Lageniform to fusoid Jug-shaped 

Basidial size ~20-31 x 7.5-15 um 30-44 x 5.5-7.8 um 
Clamp connection Present Absent 
Spheropedunculate cells Present Absent 

Rhizoid & columella Absent Present 

Locules Round Labyrinthiform 


Acknowledgments 


We express appreciation to Michael Castellano and Teresa Lebel for reviewing this 
paper. We thank Tim Flegel for reviewing English. We are grateful Ellen Bloch (curator 
of New York Botanical Garden) for the loan of Octaviania purpurea Coker & Couch 
#780569. This study was funded by BIOTEC, Thailand (TRF/BRT - The biodiversity 
Research and Training Program grant #BRT_R650001) for field survey. We appreciate 
the Department of National Parks, Wildlife and Plant Conservation, Ministry of Natural 
Resources and Environment for permitting collecting inside Khao Yai National Park. 


Octaviania violascens sp. nov. (Thailand) ... 155 


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Thailand. The Royal Institute Press, Thailand. 272 p. 

Chandrasrikul A, Suwanarit P, Sangwanit U, Morinaga T, Nishizawa Y, Murakami Y. 2008. Diversity 
of mushrooms and macrofungi in Thailand. Kasetsart University, Bangkok. 514 p. 

Coker WC, Couch JN. 1928. The gasteromycetes of the eastern United States and Canada. Chapel 
Hill The University of North Carolina Press, North Carolina. 508 p. 

Dissing H. 1963. Studies in the flora of Thailand 25: discomycetes and gasteromycetes. Dansk 
Botanisk Arkiv 23: 117-130. 

Ellingsen HJ. 1982. Some gasteromycetes from northern Thailand. Nordic Journal of Botany 2: 
283-285. http://dx.doi.org/10.1111/j.1756-1051.1982.tb01190.x 

Kornerup A, Wanscher JH. 1963. Methuen handbook of colour. Methuen & Co Ltd press, Great 
Britain. 224 p. 

Lumyong S, Sanmee R, Lumyong P, Yang ZL, Trappe JM. 2003. Mycoamaranthus cambodgensis 
comb. nov., a widely distributed sequestrate basidiomycete from Australia and southeastern 
Asia. Mycological Progress 2: 323-325. http://dx.doi.org/10.1007/s11557-006-0069-9 

Orihara T, Kasuya T, Phongpaichit S, Dissara Y. 2008. Radiigera tropica (Geastraceae, Geastrales), 
a new species from a tropical rain forest of Thailand. Mycotaxon 105: 111-117. 

Orihara T, Sawada F, Ikeda S, Yamato M, Tanaka C, Shimomura N, Hashiya M, Iwase K. 2010. 
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108-121. http://dx.doi.org/10.3852/08-168 


ISSN (print) 0093-4666 © 2012. Mycotaxon, Ltd. ISSN (online) 2154-8889 


MY COTAXON 


http://dx.doi.org/10.5248/120.157 
Volume 120, pp. 157-169 April-June 2012 


New records of cercosporoid hyphomycetes from Iran 


MAHDI PIRNIA’, RASOUL ZARE’, 
HAMID R. ZAMANIZADEH! & AKBAR KHODAPARAST? 


"Science and Research Branch, Islamic Azad University, Tehran, Iran 

*Department of Botany, Iranian Research Institute of Plant Protection, 
PO. Box 1454, Tehran, 19395, Iran 

*Department of Plant Protection, University of Guilan, Rasht, Iran 

* CORRESPONDENCE TO: pirnia@ymail.com 


ABsTRACT — Eight collections from different localities in the northern provinces of Iran 
were obtained during spring-summer 2010-11. Seven species on eight host plants were 
identified: Cercospora pantoleuca on Plantago lanceolata, Cercosporella primulae on Primula 
macrocalyx, C. virgaureae on Conyza bonariensis, Passalora bondartsevii on Medicago sp., 
Pseudocercospora danaicola on Danae racemosa, P. paraguayensis on Eucalyptus camaldulensis, 
and Scolecostigmina confluens on Crataegus melanocarpa and C. pseudomelanocarpa. ‘These 
taxa are new records to Iran. Cercosporina danaicola is recombined as Pseudocercospora 
danaicola. 


Key worps — Cercospora-like fungi, taxonomy, Asia Minor, leaf spot pathogens 


Introduction 

Cercosporoid fungi include over 30 morphologically similar anamorphic 
hyphomycetous genera that are mainly associated with leaf spots on various host 
plants such as cereals, vegetables, ornamentals, forest trees, and weeds (Crous 
& Braun 2003). Cercospora Fresen. is one of the largest genera in this group. 
In his monograph of Cercospora, Chupp (1954) listed over 1800 species names 
and accepted 1419 species. He adopted a broad generic concept and mainly 
considered the characteristics of conidia (hilum thickness, pigmentation, 
number, and arrangement). In a major taxonomic treatment, Deighton (1967, 
1973, 1974, 1976, 1979) divided and reclassified many Cercospora species into 
several segregate genera, including Cercosporella Sacc., Cercosporidium Earle, 
Paracercospora Deighton, Pseudocercospora Speg., Pseudocercosporella Deighton, 
and Pseudocercosporidium Deighton. Other morphologically similar fungi are 
members of Passalora Fr. and Scolecostigmina U. Braun. Cercospora species are 
characterized by acicular hyaline septate conidia with conspicuous hila produced 


158 ... Pirnia & al. 


on pigmented unbranched septate smooth conidiophores. Cercosporella species 
are easily distinguished from Cercospora species by colourless structures with 
thickened darkened conidiogenous loci. In Pseudocercospora, conidiogenous 
loci and hila are inconspicuous, not thickened, and not darkened. Passalora is 
distinguished by pigmented conidiophores and pigmented ellipsoid-fusiform 
to obclavate-subcylindrical conidia. Scolecostigmina is diagnosed by thick- 
walled verruculose conidiophores with conspicuous annellations in the upper 
part. Conidia in this genus are subcylindric-obclavate, smooth to verruculose, 
transversely multi-euseptate, and occasionally with a few longitudinal septa. 
Crous & Braun (2003) published an annotated checklist for Cercospora and 
Passalora names with 5720 taxa and taxonomic re-allocations of numerous 
species. Recently Braun & Crous (2007) proposed several new species, 
combinations, and names after re-examining the type collections of Cercospora 
species and other related genera. 

Iranian records of Cercospora and other related genera have been only 
poorly studied. Scharif & Ershad (1966) presented a list of fungi on various 
host plants including a few Cercospora and Pseudocercospora species. Ershad 
(1990, 2000, 2002) reported four Cercospora species from Iran and later (Ershad 
2009) corrected 59 Cercospora names previously published from Iran, partly 
according to Crous & Braun (2003), thus reducing the number of species to 22 
with 14 uncertain species. He did not follow Crous & Braun (2003) in merging 
morphologically indistinguishable taxa on various host plants in Cercospora 
apii s. lat. Recently Pirnia et al. (2010, 2012a, 2012b) studied Cercospora, 
Passalora, Ramularia, and Ramularia-like species in Iran following the species 
concepts proposed by Crous & Braun (2003) and Braun (1998). They linked 
six host plants with C. apii s. lat. Furthermore, some specimens deposited in 
the fungus reference of the Iranian Ministry of Agriculture (Iranian Research 
Institute of Plant Protection, Tehran) have been examined and proved to be 
morphologically indistinguishable from C. apii. 


Materials & methods 

Specimens with leaf spot symptoms from different localities in northern Iran 
(i.e., Guilan, Mazandaran, Golestan, northern Khorasan provinces) were collected 
during spring-summer 2010-11. Microscopic slides were prepared from stromata, 
conidiophores, and conidia in 25% lactic acid. Species were identified based on stromata 
(presence/absence) and their development, conidia (pigmentation, shape, dimensions), 
hila (thickness, darkness), conidiophores (pigmentation, dimensions) and conidial scars 
(position, thickness, darkness). Drawings were made using a drawing tube attached to 
an Olympus BH-2 microscope. 


Results 
In our study on cercosporoid hyphomycetes of Iran, we identified 70 taxa on 
100 host plants (TABLE 1). Of the 21 taxa that are new records for the mycobiota 


Cercosporoids new to Iran... 


TABLE 1. Cercosporoid hyphomycetes and hosts identified from Iran 


PATHOGEN 


*Cercospora acnidae 
C. althaeina 


*C. apii 


C. beticola 

*C. bizzozeriana 
*C. caricis 

C. cheiranthi 

C. lactucae-sativae 
*C. mercurialis 
*C. pantoleuca 
*C. peckiana 

C. sorghi 

C. traversiana 

C. violae 

C. zebrina 

C. zonata 
*Cercosporella primulae 
*C. virgaureae 
Neoovularia ovata 
Passalora bolleana 
*P. bondartsevii 

P. calotropidis 

*P. chaetomium 

P. circumscissa 

P cousiniae 

P. dubia 

P. graminis 

P. microsora 

P. personata 

P. phaeopappi 

P punctum 

P. rosae 

P rosicola 

*P. ziziphi 
Pseudocercospora abelmoschi 
P. atromarginalis 
P. cruenta 

*P. danaicola 

*P. griseola 

*P. heteromalla 

P. jujubae 

P. kaki 

P. neriella 

*P. paraguayensis 
P. punicae 


Hosts 


Amaranthus chlorostachys var. chlorostachys 
*Gossypium hirsutum 


Abutilon theophrasti, Euphorbia heterophylla, Gerbera jamesonii, 
Medicago sp., Pelargonium zonale, Petunia hybrida, Solanum 
lycopersicum, Vigna sinensis, Zantedeschia aethiopica 

Beta vulgaris, B. maritima 

Cardaria draba 

Carex orbicularis 

Cheiranthus cheiri 

Lactuca sativa, *L. serriola 

Mercurialis annua 

Plantago lanceolata 

Rumex sanguineus, R. crispus 

*Sorghum halepense 

Trigonella foenum-graecum 

Viola sylvestris, Viola sp. 

Medicago sp. 

Vicia faba 

Primula macrocalyx 

Conyza bonariensis 

Salvia hypoleuca, S. limbata, S. nemorosa 

Ficus carica 

* Medicago sp. 

Calotropis procera 

Euphorbia marschalliana 

Cerasus vulgaris, Prunus domestica 

Cousinia sp. 

*Chenopodium album 

*Agropyron sp., *Poa annua, * Stipa sp. 

Tilia begoniifolia 

Arachis hypogaea 

Phaeopappus aucheri 

Anethum graveolens, Foeniculum vulgare, Petroselinum sativum 

*Rosa persica, Rosa sp. 

Rosa sp. 

Ziziphus spina-christi 

Hibiscus cannabinus 

Solanum nigrum 

Vigna sinensis 

Danae racemosa 

Phaseolus vulgaris 

Rubus sp. 

Ziziphus spina-christi 

Diospyros lotus, D. kaki 

Nerium oleander 

Eucalyptus camaldulensis 

Punica granatum 


159 


160 ... Pirnia & al. 


Table 1, concluded 


PATHOGEN 

P. rubi 

*P. salicina 

P. salvadorae 

P. sphaerellae-eugeniae 

P vitis 

Ramularia anchusae 

R. brunnea 

R. cynarae 

R. geranii var. geranti 

R. grevilleana var. grevilleana 
R. heraclei 

*R. inaequalis 

R. lamii var. lamii 

*R. macularis 

*R. pratensis var. pratensis 
R. rhabdospora 

R. rubella 

R. rumicis 

R. sambucina 

R. simplex 

R. uredinicola 

R. urticae 

Ramulariopsis gossypii 
*Scolecostigmina confluens 
Sirosporium celtidis 


Hosts 

Rubus sp. 

Salix alba 

Salvadora persica 

Eugenia jambos 

Vitis sylvestris, V. vinifera 

Anchusa italica, A. ovata 

Tussilago farfara 

Carthamus oxyacantha, C. tinctorius 
Geranium pyrenaicum 

Fragaria x ananassa, Potentilla reptans 
Heracleum persicum 

*Calendula persica 

*Mentha piperita, Mentha sp. 
Chenopodium album 

Rumex crispus, Rumex sp. 

Plantago lanceolata 

*Rumex conglomeratus 

Rumex crispus 

Sambucus ebulus 

Ranunculus oxyspermus, R. sahendicus 
Uredinia of Melampsora sp. on Salix babylonica 
Urtica dioica, Urtica urens 

Gossypium hirsutum 

Crataegus melanocarpa, C. pseudomelanocarpa 
Celtis australis 


* = new pathogen and host records 


of Iran, seven are treated in detail in this paper. All specimens are deposited in 
the fungus reference of Iranian Ministry of Agriculture “IRAN” at the Iranian 
Research Institute of Plant Protection. Synonyms are listed in Crous & Braun 
(2003) and Braun (1995, 1998). 


Taxonomy 


Cercospora pantoleuca Sacc., Michelia 1: 268 (1878) Fic. 1 

Leaf spots circular, numerous, brown to blackish brown with gray center, 
1-3 mm in diameter; caespituli amphigenous, mostly epiphyllous, punctiform; 
stromata small to fairly prominent, brown, 20-25 um wide; conidiophores 
fasciculate, 8-15 stalks, arising through stomata, pale olivaceous-brown, paler 
towards the tip, aseptate or with few inconspicuous septa, straight, subcylindrical 
to geniculate-sinuous, not branched, smooth, thin, 15-50 x 2.5-5 um; conidial 
scars conspicuous, thickened and darkened, terminal and lateral, 1.5-2 um 
wide; conidia formed singly, hyaline, acicular, narrowly obclavate, straight to 


Cercosporoids new to Iran... 161 


Fic. 1. Cercospora pantoleuca on Plantago lanceolata. 
A. Conidiophores, B. Conidia, C. Symptoms on leaf (Bar = 50 um). 


slightly curved, smooth, thin, 3-7-septate, base truncate, tip obtuse to subacute, 
40-90 x 2.5-4 (-5) um; hilum thickened and darkened. 
SPECIMEN EXAMINED: IRAN, NORTHERN KHORASAN PROVINCE, Shirvan, on Plantago 
lanceolata L., 3 July 2011, B. Bicharanlou (IRAN 15498 F). 
Note—This species is characterized by short conidiophores and conidia. 
Conidiophores are pale olivaceous-brown to subhyaline, and conidial scars 
are conspicuous, terminal, and lateral. These characters distinguish this 
species from other Cercospora species. The Iranian specimen examined closely 
resembles Braun's (1995) description of this species but slightly differs in having 
faintly pigmented conidiophores. 


Cercosporella primulae Allesch., Ber. Bayer. Bot. Ges. 2: 18 (1892) Fic. 2 

Leaf spots circular to subcircular, yellowish, ochraceous to brown, margin 
indefinite, surrounded by a yellowish halo, 2-8 mm in diameter; caespituli 
amphigenous, punctiform, whitish; stromata substomatal to intraepidermal, 
composed of few swollen hyphal cells, colourless; conidiophores in small 
fascicles, 3-5 stalks, rarely solitary, arising through stomata or erumpent through 
the cuticle, hyaline, straight to geniculate-sinuous, not branched, smooth, thin- 


162 ... Pirnia & al. 


A B 


Fic. 2. Cercosporella primulae on Primula macrocalyx. 
A. Conidiophores, B. Conidia, C. Symptoms on leaf (Bar = 50 um). 


walled, 0-1-septate, 20-45 x 2.5-5 um; conidial scars conspicuous, thickened; 
conidia formed singly, hyaline, obclavate-acicular to subcylindrical, smooth, 
thin, indistinctly multiseptate, 0-7-septate, base truncate to obconicallytruncate, 
tip subacute, 20-85 x 3-5 um; hilum slightly thickened and darkened. 
SPECIMEN EXAMINED: IRAN, GOLESTAN PROVINCE, Gorgan, on Primula macrocalyx 
Bunge, 13 May 2011, M. Pirnia (IRAN 15479 F). 
Note—This taxon is characterized by moderately short and_ hyaline 
conidiophores and conidia. Because of its hyaline conidiophores and conidia 
as well as the structure of the scars, Braun (1995) maintained this species in 
Cercosporella. 


Cercosporella virgaureae (Thiim.) Allesch., Hedwigia 34: 286 (1895) Fic. 3 

Leaf spots subcircular to irregular, at first yellowish brown, later grayish 
brown, 2-6 mm in diameter; caespituli hypophyllous, whitish to grayish white, 
punctiform to dense; stromata substomatal, composed of some aggregated 
swollen hyphal cells, conidiophores fasciculate, 3-8 stalks, arising from 
stromata, emerging through stomata, hyaline, below straight, geniculate-sinuous 
towards the apex, not branched, 0-1-septate, 25-65 x 4-8 um; conidiogenous 
cells integrated, terminal; conidial scars conspicuous, thickened and darkened, 
terminal and lateral; conidia formed singly, hyaline, subcylindrical to obclavate, 


Cercosporoids new to Iran ... 163 


Fic. 3. Cercosporella virgaureae on Conyza bonariensis. 
A. Conidiophores, B. Conidia, C. Symptoms on leaf (Bar = 50 um). 


straight to slightly curved, smooth, thin, multiseptate, 3-7 septa, base truncate 
to obconically truncate, tip mostly obtuse, 40-95 x 4-7 um; hilum distinct. 
SPECIMEN EXAMINED: IRAN, GUILAN PROVINCE, Lahijan, on Conyza bonariensis (L.) 
Cronquist, 29 Nov. 2010, M.R. Mirzaee (IRAN 15480 F). 
Note—Morphology of the specimen examined agrees with the description 
provided by Braun (1995). Cercosporella virgaureae is widespread and has a 
wide host range on many genera of Asteraceae. This taxon is well characterized 
by hyaline to subhyaline structures, long conidiophores and conidia, and 
numerous conspicuous conidial scars on conidiophores. 


Passalora bondartsevii U. Braun & Melnik, Trudy Bot. Inst. im. V.L. Komarova 20: 
43 (1997) Fig. 4 
Leaf spots circular to irregular, pale brown, surrounded by a yellowish 
halo, 5-10 mm in diameter; caespituli epiphyllous, punctiform; conidiophores 
in small to dense fascicles, 7-20 stalks, arising from stromata, brown, erect, 
subcylindrical, slightly geniculate-sinuous towards the apex, smooth, thin, 
continuous to septate, sometimes constricted at the septa, 40-90 x 4-5 um; 


164 ... Pirnia & al. 


Fic. 4. Passalora bondartsevii on Medicago sp. 
A. Conidiophores, B. Conidia, C. Symptoms on leaf (Bar = 50 um). 


conidiogenous cells integrated, terminal; conidial scars moderately thickened 
and darkened; conidia formed singly, olivaceous-brown, ellipsoid, obovoid, 
smooth to rough, 0-3-septate, base obconically truncate, apex obtuse to rounded, 
10-20 x 5-7.5 um; hilum darkened, unthickened to slightly thickened. 

SPECIMEN EXAMINED: IRAN, MAZANDARAN PROVINCE, Babolsar, on Medicago sp., 25 

June 2011, M. Rafiee (IRAN 15499 F). 
Note—This taxon was originally established by Braun & Melnik (1997) 
on Onobrychis vicifolia Scop., from Russia and is only known from the type 
collection. Passolora bondartsevii is diagnosed by ellipsoid-obovoid to 
obclavate-subcylindrical, smooth to faintly rough conidia. In our specimen 
the conidia are ellipsoid, obovoid, and mostly rough, but we did not see any 
obclavate-subcylindrical conidia. However, other characters agree well with 
the description in Braun & Melnik (1997). Therefore, we assign the Iranian 
collection tentatively to P bondartsevii even though another host genus is 
involved. 


Pseudocercospora danaicola (Vienn.-Bourg.) Pirnia & Zare, comb. nov. Fic. 5 
MycoBank MB 563391 


= Cercosporina danaicola Vienn.-Bourg. Ann. Phytopathol. 
2(4): 689 734 (1971 [“1970”]) as “danaecola”. 


Leaf spots circular to subcircular, sometimes with spacious blight specially at 
the tip of leaves, reddish brown, 10-15 mm in diameter; caespituli amphigenous, 


Cercosporoids new to Iran... 165 


punctiform; stromata present, globular, well developed, dark brown, 45-60 um 
wide; conidiophores in dense fascicles, arising from stromata, pale olivaceous- 
brown, subcylindrical, sinuous, rarely once geniculate, not branched, smooth, 
thin, aseptate, 8-30 x 2-3 um; conidiogenous loci inconspicuous; conidial 
scars unthickened and inconspicuous; conidia formed singly, pale olivaceous, 
acicular, straight to slightly curved, smooth, thin, indistinctly 1-5-septate, base 
truncate, tip subacute, 65-130 x 2.5-4 um; hilum inconspicuous, unthickened 
and not darkened. 

SPECIMENS EXAMINED (deposited as “Cercosporina danaecola”): IRAN, GOLESTAN 

PROVINCE, Khan-bebin, Shirabad forest, on Danae racemosa Moench, 7 May 1976, 

D. Ershad (IRAN 515 F); MAZANDARAN PROVINCE, Nowshahr, on D. racemosa, 19 

Aug. 1973, collector unknown (IRAN 516 §, neotype, designated here). 
Note—The name Cercosporina danaicola was originally published by Viennot- 
Bourgin based on material from Iran (Viennot-Bourgin et al. 1971). Careful 
microscopic examination of morphological characteristics showed that the 
conidiogenous loci and hila are inconspicuous, unthickened, and not darkened. 
Crous & Braun (2003) place species with these morphological features into 
Pseudocercospora. Type material could not be traced, but based on the Iranian 


Fic. 5. Pseudocercospora danaicola on Danae racemosa. 
A. Conidiophores, B. Conidia, C. Symptoms on leaf (Bar = 50 um). 


166 ... Pirnia & al. 


specimens examined, this species can be transferred to Pseudocercospora. The 
collection IRAN 516 is designated as neotype. 


Fic. 6. Pseudocercospora paraguayensis on Eucalyptus camaldulensis. 
A. Conidiophores, B. Conidia, C. Symptoms on leaf (Bar = 50 um). 


Pseudocercospora paraguayensis (Tak. Kobay.) Crous, Mycotaxon 57: 270 (1996) 
Fic. 6 
Leaf spots irregular, vein-limited, pale brown to dark grayish brown, border 
slightly raised, 2-5 mm in diameter, sometimes coalescing into a large blotch; 
caespituli amphigenous, mostly hypophyllous, punctiform; stromata present, 
well developed, brown, 50-70 um wide; conidiophores densely fasciculate, 
compact, subhyaline to pale olivaceous, subcylindrical, aseptate, not branched, 
sinuous to rarely once geniculate, straight to curved, rounded at the apex, 
10-30 x 2-3 um; conidial scars unthickened and inconspicuous; conidia 
formed singly, subhyaline or very pale olivaceous, narrowly obclavate, straight 
to curved, indistinctly 2-6-septate, apex subacute or subobtuse, base truncate 
to obconically truncate, 22-70 x 2-3 um; hilum inconspicuous, unthickened 
and not darkened. 
SPECIMEN EXAMINED: IRAN, GOLESTAN PROVINCE, Shastkola forest, on Eucalyptus 
camaldulensis Dehnh., 8 Nov. 2010, M. Pirnia & R. Zare (IRAN 15481 F). 
Note—Kobayashi introduced Cercospora paraguayensis on Eucalyptus sp. from 
Paraguay (see Crous 1998). 


Cercosporoids new to Iran... 167 


Fic. 7. Scolecostigmina confluens on Crataegus pseudomelanocarpa. 
A. Conidiophores, B. Conidia, C. Symptoms on leaf (Bar = 50 um). 


Scolecostigmina confluens (Lieneman) U. Braun, New Zealand J. Bot. 37: 325 (1999) 
Fic. 7 
Leaf spots irregular, dark brown, scattered on leaf surface, but mostly at 
the tip and edge of leaves, 5-15 mm in diameter; caespituli amphigenous, 
mostly epiphyllous, punctiform; stromata prominent, subglobose, dark brown, 
30-65 um wide; conidiophores in dense fascicles, 15-30 stalks, arising from 
stromata, dark brown, paler and attenuated towards the apex, 0-1-septate, 
cylindrical, straight to curved, sinuous to rarely once geniculate in the upper 
part, not branched, verruculose, thick-walled, 25-75 x 4-7 um; conidiogenous 
cells integrated, terminal, proliferation percurrent, annellate; conidia solitary, 
dark brown, paler and narrower towards the tip, subcylindrical-obclavate, 
slightly curved, verrucose, thick-walled, transversely pluriseptate, occasionally 
with few longitudinal or oblique septa, base truncate, tip subacute, 75-140 x 
4—8 um. 
SPECIMENS EXAMINED: IRAN, GOLESTAN PROVINCE, Ghorogh forest park, on Crataegus 
pseudomelanocarpa Popov ex Lincz. 14 Sep. 2010, M. Pirnia (IRAN 15472 F); GUILAN 
PROVINCE, Rasht, Saravan, on C. melanocarpa M. Bieb., 27 July 2007, A. Khodaparast 
(IRAN 15473 F). 
Note—The genus Scolecostigmina was originally described as having thick- 
walled verruculose conidiophores with conspicuous annellations and conidia 


168 ... Pirnia & al. 


that are subcylindrical-obclavate, smooth to verruculose, transversely multi- 
euseptate conidia, and occasionally with a few longitudinal septa (Braun et al. 
1999). 


Acknowledgments 

The authors are grateful to U. Braun (Martin-Luther-Universitat, Halle, Saale, 
Germany) for providing part of his work on cercosporoid hyphomycetes from Russia as 
well as his pre-submission review. A.J.L. Phillips (Universidade Nova de Lisboa, Portugal) 
is thanked for preliminary review of the manuscript. We also thank S. Pennycook for 
improving the text, particularly the nomenclature. A.R. Ghorbanian (Damghan Branch, 
Islamic Azad University, Iran) and B. Djavadi (Iranian Research Institute of Plant 
Protection, Iran) are thanked for identifications of host plants. 


Literature cited 

Braun U. 1995. A monograph of Cercosporella, Ramularia and allied genera (phytopathogenic 
hyphomycetes), Vol. 1. IHW-Verlag, Eching. 

Braun U. 1998. A monograph of Cercosporella, Ramularia and allied genera (phytopathogenic 
hyphomycetes), Vol. 2. IHW-Verlag, Eching. 

Braun U, Crous PW. 2007. The diversity of cercosporoid hyphomycetes: new species, combinations, 
names and nomenclatural clarifications. Fungal Diversity 26: 55-72. 

Braun U, Melnik VA. 1997. Cercosporoid fungi from Russia and adjacent countries. Trudy Bot. 
Inst. im. V.L. Komarova (St. Petersburg) 20: 1-130. 

Braun U, Mouchacca J, McKenzie EHC. 1999. Cercosporoid hyphomycetes from New Caledonia 
and some other South Pacific islands. New Zealand Journal of Botany 37: 297-327. 
http://dx.doi.org/10.1080/0028825X.1999.9512636 

Chupp C. 1954. A monograph of the fungus genus Cercospora. Ithaca, New York. Published by the 
author. 

Crous PW. 1998. Mycosphaerella spp. and their anamorphs associated with leaf spot diseases of 
Eucalyptus. Mycologia Memoirs 21: 1-170. 

Crous PW, Braun U. 2003. Mycosphaerella and its anamorphs: 1. Names published in Cercospora 
and Passalora. CBS Biodiversity Series 1: 1-569. 

Deighton FC. 1967. Studies on Cercospora and allied genera. II. Passalora, Cercosporidium and 
some species of Fusicladium on Euphorbia. Mycological Papers 112: 1-80. 

Deighton FC. 1973. Studies on Cercospora and allied genera. IV. Cercosporella Sacc., 
Pseudocercosporella gen. nov. and Pseudocercosporidium gen. nov. Mycological Papers 133: 
1-62. 

Deighton FC. 1974. Studies on Cercospora and allied genera. V. Mycovellosiella Rangel and a new 
species of Ramulariopsis. Mycological Papers 137: 1-75. 

Deighton FC. 1976. Studies on Cercospora and allied genera. VI. Pseudocercospora Speg., Pantospora 
Cif. and Cercoseptoria Petr. Mycological Papers 140: 1-168. 

Deighton FC. 1979. Studies on Cercospora and allied genera. VII. New species and redispositions. 
Mycological Papers 144: 1-56. 

Ershad D. 1990. New records of two Cercospora species for Iran. Iranian Journal of Plant Pathology 
26: 41. 

Ershad D. 2000. Two new species of mitosporic fungi. Rostaniha 1: 1-9. 

Ershad D. 2002. A new Cercospora species from Iran. Rostaniha 3: 47-50. 


Cercosporoids new to Iran... 169 


Ershad D. 2009. Fungi of Iran. Iranian Research Institute of Plant Protection, Tehran. 

Pirnia M, Zare R, Zamanizadeh HR, Khodaparast A. 2010. Contribution to the identification of 
Cercospora species in Iran. Rostaniha 11(2): 183-189. 

Pirnia M, Zare R, Zamanizadeh HR, Khodaparast A, Javadi Estahbanati AR. 2012a. Contribution 
to the identification of the genus Passalora in Iran. Applied Entomology and Phytopathology 
80 (1) (in press). 

Pirnia M, Zare R, Zamanizadeh HR, Khodaparast A, Javadi Estahbanati AR. 2012b. Taxonomic 
study of the genus Ramularia and Ramularia-like genera in Iran. Rostaniha 13(1) (in press). 

Scharif G, Ershad D. 1966. A list of fungi on cultivated plants, shrubs and trees of Iran. Ministry of 
Agriculture, Plant Pests and Diseases Research Institute, Evin, Tehran. 

Viennot-Bourgin G, Ale-Agha N, Ershad D. 1971 [“1970”]. Les champignons parasites de I’Iran. 
(Nouvelle contribution). Annales de Phyopathologie 2(4): 689-734. 


ISSN (print) 0093-4666 © 2012. Mycotaxon, Ltd. ISSN (online) 2154-8889 


MY COTAXON 


http://dx.doi.org/10.5248/120.171 
Volume 120, pp. 171-179 April-June 2012 


Phaeocollybia nigripes (Agaricomycetes), a new species from Brazil 


VICTOR R.M. COIMBRA”, TATIANA B. GIBERTONI! & FELIPE WARTCHOW? 


' Universidade Federal de Pernambuco, Departamento de Micologia/CCB, 
Av. Prof. Nelson Chaves, s/n, CEP: 50670-901, Recife, PE, Brazil 

? Universidade Federal da Paraiba, Departamento de Departamento de Sistematica e Ecologia, CEP: 
58051-970, Jodo Pessoa, PB, Brazil 


* CORRESPONDENCE TO: vick_mat_coimbra@yahoo.com. br 


ABSTRACT —Phaeocollybia nigripes is described from an Atlantic Forest fragment in 
Pernambuco, Northeast Brazil. It is characterized by a stipe that is uniformly slender with a 
slightly swollen base and dark grayish brown below to almost black at the apex, brown pileus, 
large basidiospores that average 12.4 x 6.6 um, hyphoid to narrowly clavate cheilocystidia, 
and abundant clamp connections. The holotype of P megalospora var. megalospora, the 
putatively most similar taxon, was examined. A key to the six taxa reported from Brazil is 
also presented. 


Key worps — Agaricales, Hymenogastraceae, neotropic, taxonomy 


Introduction 

Phaeocollybia R. Heim was originally described to accommodate taxa 
among the brown-spored Agaricales with smooth and moist to viscid pilei and 
long pseudorhizas (Heim 1931). Matheny et al. (2006), based on multilocus 
phylogenetic analyses, placed the genus as represented by P festiva (Fr.) 
R. Heim in the Hymenogastraceae clade, but Kirk et al. (2008), continued to 
refer Phaeocollybia to Cortinariaceae. More recently, Petersen et al. (2010), also 
using molecular data, treated P festiva and P dissiliens A.H. Sm. & Trappe in the 
bolbitioid clade with Bolbitius Fr., Panaeolus (Fr.) Quél., and Conocybe Fayod. 

Phaeocollybia has been rarely reported in Brazil. The few taxa known from 
this country were all reported from the Amazon region by Singer (1987), 
who cited P brasiliensis .A. Aguiar ex Singer, P. elaeophylla Singer, P. flava 
I.A. Aguiar ex Singer, P megalospora I1.A. Aguiar ex Singer var. megalospora, 
and P. megalospora var. tetraspora Singer. 

In this paper, we describe a new species based on a collection from the 
Atlantic Forest of Pernambuco, Northeast Brazil, and provide a key to the 
Brazilian Phaeocollybia species. 


172 ... Coimbra, Gibertoni & Wartchow 


Materials & methods 

One basidioma was collected in an Atlantic Forest fragment called Ecological 
Reserve of Dois Irmaos (8°01'15"S 34°52'30"W), a 387.4 ha area comprising a remnant 
of Atlantic Forest and a public zoo in the urban perimeter of the municipality of Recife 
(Ribeiro et al. 2007, Souza et al. 2009). 

Colors are coded according to Watling (1969). The key to species is based mainly on 
data given by Singer (1987). Basidiospore data terminology follows Tulloss et al. (1992) 
but based on 25 basidiospores measured from a single basidioma (Wartchow 2009). 
Abbreviations include L(W) = basidiospore length (width) average, Q = the length : 
width ratio range as determined from all measured basidiospores, and Q = the Q value 
averaged from all basidiospores measured. 

Scanning electron microscopy (SEM) studies were conducted at the “Laboratorio de 
Microscopia Eletrénica’ (DF/UFPE). Sections were removed from dried basidiomata 
and mounted directly on aluminum stubs using carbon adhesive tabs. The fragments 
were coated with 8-13 nm of gold using a sputter coater and examined with a scanning 
electron microscope. 


Phaeocollybia nigripes Wartchow & V. Coimbra sp. nov. FIGs. 1-6, 8 
MycoBank MB 518815 
Ab Phaeocollybia megalospora var. megalospora differt stipe coloribus apice niger versus 
fuscus basin, cheilocystidiis tenui clavati vel cylindraceous et catenatus, et pileipellis 
hipodermio ex elementis pro ratione voluminosis. 
Ho torype: Brazil, Pernambuco, Recife, Ecological Reserve of Dois Irmaos, 10.iii.2009 
V.R.M. Coimbra, J.M. Baltazar & L. Trierveiler-Pereira s.n. (URM 82277, holotype). 


Erymo oy: Latin for ‘black foot; in reference to the stipe color. 


PiLEus 11 x 10 mm, conic, reddish brown to dark brown (“Date brown 24’), 
surface smooth, margin slightly short-sulcate and involute. LAMELLAE adnexed, 
close, lamellulae present, dark grayish brown (slightly lighter than “Drab 33’) 
with dark gray edges (under 10x lens), edges entire. Veil not evident. STIPE 70 
x 1.5-2 mm (upper portion apart from pseudorhiza 55 mm long), cylindrical 
above a swollen base, central, dark brown (‘Cigar brown 16’), shiny metallic 
graphite-black at apex with a vinaceous base under intense light, surface 
smooth, hollow. Pseudorhiza thin, spiraling, % of the stipe length. 
BASIDIOSPORES (10—)10.8-14.5(-15) x (5.5-)6-7.3(-8) um (L = 12.4 um; W 
= 6.6 um; Q = (1.69-)1.77-2.00(-2.29); Q = 1.88), amygdaliform to sometimes 
limoniform, thin-walled, surface minutely verrucose, brown. BAsip1a 24-33 x 
9-11 um, clavate, thin-walled, with prominent sterigmata, predominantly 2- 
sterigmate. PLEUROCYSTIDIA absent. CHEILOCYSTIDIA (13—)16-25 x 5-6 um, 
narrowly clavate to hyphoid (located in terminal position of chains of short 
celled hyphae), forming dense tufts on the sterile edge, thin-walled, hyaline. 
TIBIIFORM DIVERTICULA abundant on pseudorrhizal pellis, 11-32 x 1.8-3 um, 
narrowly lanceolate to subcapitate, sometimes with apical droplet, some with 
small lateral projections, hyaline, colorless, thin walled; on stipe more scattered 


Phaeocollybia nigripes sp. nov. (Brazil) ... 173 


Fic. 1. Phaeocollybia nigripes (holotype). Basidioma. Scale bar = 10 mm. 


and slightly smaller 11-16 x 2-3 um, hyaline, thin walled. PILEIPELLIs with 
periclinal to sometimes anticlinal hyphae of 3-6 um wide, pigments encrusting, 
yellowish brown, showing some gelatinized zones; hypodermium somewhat 
pseudoparenchymatous, elements 20-27 x 13-18 um, yellowish-brown. 
LAMELLAR TRAMA regular and parallel, hyphae ranging 4.5-15.5 um, thin- 
walled. CLAMP CONNECTIONS abundant. 

ECOLOGY & DISTRIBUTION: Solitary on soil under dicotyledonous trees, 
including representatives of the ectomycorrhizal families (Smith & Read 2008) 
Caesalpiniaceae [Senna Mill., Chamaecrista (L.) Moench, Dialium L., Hymenaea 
L., Sclerolobium Vogel, Zollernia Maximil. & Nees], Nyctaginaceae (Boerhavia 


174 ... Coimbra, Gibertoni & Wartchow 


5 


Fics. 2-6. Phaeocollybia nigripes (holotype): 2. Basidioma. 3. Basidiospores. 4. Basidia. 5. Hyphal 
elements of the lamella edge. 6. Pileipellis and adjacent inflated elements of the hypodermium. 
Fic. 7. P. megalospora var. megalospora (holotype): Pileipellis and adjacent hyphae of the 
hypodermium. Scale bars: 2 = 10 mm; 3-7 = 10 um. 


L., Guapira Aubl.) and Polygonaceae (Coccoloba P. Browne). Known only from 
the type locality in the Atlantic Forest of Northeast Brazil. 

ADDITIONAL SPECIMEN EXAMINED: Phaeocollybia megalospora var. megalospora— 

BRAZIL. Amazonas, road Manaus-Caracarai km 45, 11.vii.1977, R. Singer & I. Araujo, 

Singer B 10082 (INPA 102697, holotype!). 
REMARKS: The distinctive microscopic features of P nigripes include 
basidiospores longer than 11 um, predominantly 2-spored basidia, cylindric- 
hyphoid to narrowly clavate cheilocystidia, anda slightly pseudoparenchymatous 
hypodermium. The pseudorhiza of our new species was broken during 
collection. 

Following the classification proposed by Bandala & Montoya (1994), 

P. nigripes would be referred to subgen. Fibulophaeocollybia Bandala & Montoya 
sect. Subattenuatae Singer based on the presence of clamp connections (most 
easily seen at the cheilocystidial bases) and limoniform basidiospores longer 
than 7 um (Bandala & Montoya 1994). Norvell & Exeter (2009), who felt that 
infrageneric classification is premature in the absence of molecular analyses of 
the whole genus, suggested that cheilocystidial morphology might be a better 


Phaeocollybia nigripes sp. nov. (Brazil) ... 175 


» - 


\ = 


isk “6; BEE 


13kU ~6, BEB Zhim 34 29 


Fics. 8-9. SEM of basidiospores (photos by Sérgio Santos). 
8. Phaeocollybia nigripes. 9. P. megalospora. 


176 ... Coimbra, Gibertoni & Wartchow 


morphological character for identifying monophyletic groups below genus 
level in Phaeocollybia. 

Phaeocollybia megalospora var. megalospora, characterized by Singer (1987) 
as also producing 2-sterigmate basidia and similar sized basidiospores, differs 
in its yellowish to brownish stipe, pileipellis with a gelatinized ixocutis and 
cutiform rather than pseudoparenchymatous hypodermium, ampullaceous 
cheilocystidia, and more strongly verrucose basidiospores (Singer 1987). We 
examined the holotype (Fics. 7, 9) of P megalospora, which we believe is the 
same basidioma depicted in Singer (1987: Plate II, fig. 5), and noted that the 
stipe was apparently broken and lacked the pseudorhiza (which typically bears 
abundant tibiiform diverticula according to Norvell 1998), so that only one 
very small (<7 x 2 um) tibiiform diverticulum was observed. The pileipellis 
comprised gelatinized erect, non-incrusted, 1-3.5 um wide, yellowish hyphae 
with the hypodermium comprising hyphae <5 um wide, radially oriented, 
and with dark brown vacuolar pigments (Fig. 7). The basidiospores are very 
similar in size 11-15 x (6-)6.5-8 um (L = 12.9 um; W = 7.2 um; Q = (1.43-) 
1.50-2.00(-2.14); Q = 1.80) but more strongly verrucose (Fic. 9) than those 
found in our species, supporting our conclusion that P megalospora var. 
megalospora and P. nigripes are distinct species. 

Another taxon with blackish tints in the basidioma is P singularis E. Horak & 
Halling from Colombia (Horak & Halling 1991) and the Talamanca Mountains 
in Costa Rica (Norvell pers. corr.), but it obviously differs in the blackish 
lavender pileus, violaceous stipe, smaller (8-9.5 x 4.5-5 um) basidiospores, and 
tibiiform cheilocystidia. 

Phaeocollybia phaeogaleroides Norvell is another similar species with 
relatively large basidiospores. It shares with P. nigripes the dark brown pileus 
and stipe, clamp connections, and cylindrical cheilocystidia (Norvell 2002) but 
differs in the hygrophanous pileus, shorter narrower basidiospores 9-12(-13) 
x 5-6.5 um (L = 10 um; W = 5.8 um), 4-spored basidia, and longer (< 80 um) 
subcapitate cheilocystidia (Norvell 2002). 

Phaeocollybia longistipitata Halling & E. Horak, P odorata E. Horak, 
P. pleurocystidiata Norvell & Redhead, P. singeri Guzman et al., and P. tentaculata 
E. Horak are other slender stiped taxa that are segregated primarily by their 
smaller basidiospores and lack of clamp connections (Horak 1977, Bandala et 
al. 1989, Norvell & Redhead 2000, Halling & Horak 2008). 

Phaeocollybia columbiana Singer from tropical rain forest of Colombia also 
shares relatively large (10-12 x 6-7 um) basidiospores. However, it differs in 
the spadiceous pileus with whitish centre, ochraceous brown stipe, ventricose 
cheilocystidia, and absence of clamp connections (Singer 1970, Horak 1977). 

Phaeocollybia amazonica Singer, from the Bolivian Amazon, differs from 
P. nigripes macroscopically in pileus size (40-45 mm diam), ochraceous brown 


Phaeocollybia nigripes sp. nov. (Brazil) ... 177 


to rusty lamellae, and cinnamon stipe and microscopically in basidiospore 
shape and larger (>30 x 6 um) cheilocystidia (Singer 1961, 1970). 

Other lowland Amazonian Brazilian taxa — P. brasiliensis, P. elaeophylla, 
P. flava, P. megalospora var. tetraspora — are easily segregated by their smaller 
basidiospores (Singer 1987). 

Here we also address the ecological status of Phaeocollybia nigripes. Earlier 
studies implied Phaeocollybia as saprotrophic with the pseudorhizae arising 
from buried wood (Smith 1957; Smith & Trappe 1972). Subsequently Redhead 
& Malloch (1986), who studied the temperate P christinae (Fr.) R. Heim and 
P. jennyae (P. Karst.) Romagn., concluded that Phaeocollybia is parasitic based 
on the successful excavation of a pseudorhiza connected to a senescent spruce 
rootlet. Norvell (1998), demonstrating that P. redheadii Norvell, P. kauffmanii 
(A.H. Sm.) Singer, and several other taxa present ectomycorrhizal mantles 
and Hartig-net in western hemlock and noble fir roots, emphasized that all 
Pacific Northwest North American Phaeocollybia species are found in forests 
with Tsuga, Picea, Abies, Pseudotsuga, Lithocarpus, Quercus, and/or Pinus, all 
ectomycorrhizal-forming genera (Smith & Read 2008). Most recently, Norvell 
& Exeter (2009) cite the presence of Hartig-net and high nitrogen isotope 
readings presented by the studied species as the main characters supporting 
the ectomycorrhizal nature of Phaeocollybia in the Pacific Northwest. 

Neotropical phaeocollybias from Colombia and Costa Rica occur mostly 
under the ectomycorrhizal tree genus Quercus (Singer 1987, Horak & Halling 
1991, Halling & Horak 2008), while others from lowland Amazon forests 
were found in “terra firme,’ a typical anectotrophic forest type according to 
Singer & Aratijo (1979). In the case of PB nigripes, although no ectomycorrhizal 
connection was detected, representatives of Caesalpiniaceae, Nyctaginaceae, and 
Polygonaceae occurred in the collection area (Guedes 1998, Ferreira et al. 2007, 
Souza et al. 2009); these families include genera shown to have ectomycorrhizal 
associations in the tropical forests of Ecuador: e.g., Coccoloba (Polygonaceae) 
and Guapira (Nyctaginaceae) (Tedersoo et al. 2010). 


Artificial key for the Brazilian taxa of Phaeocollybia 


1. Basidiospores small, 5-5.5 x 4.5-5 um; pileus yellow..................00. P. flava 
1. Basidiospores longer, > 7 um long; pileus with brown tints...................00. 2 
2. Basidiospores <10-15 um long; basidia mostly with two sterigmata .............. 3 
2. Basidiospores with largest length not longer than 11.5 um; basidia with four 
sterigmata (in P elaeophylla 1-4 sterigmata) ............ 0... cece eee ee eee -: 


3. Stipe yellowish to brownish towards the base; cheilocystidia ampullaceous; 
hypodermium cutiform made of hyphae 3-9 um wide 
ET EE EN EE P. megalospora var. megalospora 


178 ... Coimbra, Gibertoni & Wartchow 


3°. Stipe dark brown with shiny metallic graphite-black apex; cheilocystidia narrowly 
clavate to hyphoid (located in terminal position of chains of short-celled 
hyphae); hypodermium pseudoparenchymatous made of elements of 


DORD FPN SVS I. es Lele an beg avn, thy Bawa -ap g Monk hd Manny 4 Banka g Beawave gg avk's P. nigripes 
4, Lamellae olive; basidiospores 8.5-10.3 x 4.5-6.5 um, cheilocystidia 

heteromorphous 17-49 x 3.5-5.5 um, sometimes tibiiform-like .... P elaeophylla 
4 Searivellae* Wak eTiG OHVecCOlO = (Liste sedi! les de, Ret ae, a EM ath aed eset pli 5 


5. Pileus striate; basidiospores (7—)7.5-8.3 x 4.7-5.5 um; cheilocystidia ventricose 


frequently capitate (tibiiform?) 25-32 x 6-7.5 um .............. P. brasiliensis 
5. Pileus not striate; basidiospores 9.5-11.5 x 6-7 um, cheilocystidia clavate, 
cylindrical or utriform of 40-50 x 3.8-6.5um .... P megalospora var. tetraspora 


Acknowledgments 

The authors would like to thank Dr. Lorelei L. Norvell for patiently reviewing all 
previous manuscript drafts and teaching about this interesting genus. Dr. Clark L. 
Ovrebo is also kindly acknowledged for reviewing our article. M.Sc. Juliano M. Baltazar 
and M.Sc. Larissa Trierveiler-Pereira are thanked for help during the field trips, FACEPE 
for providing a post-doctoral grant to FW (BFP 0100-2.03/09) and scholarship to VRMC 
(BIC 0061-2.03/08) and partially financing this research (APQ 0444-2.03/08). Sergio 
Santos, of the Department of Physics (Universidade Federal de Pernambuco, Brazil) 
is acknowledged for the scanning electron micrographs of the basidiospores of both 
species. 


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ISSN (print) 0093-4666 © 2012. Mycotaxon, Ltd. ISSN (online) 2154-8889 


MY COTAXON 


http://dx.doi.org/10.5248/120.181 
Volume 120, pp. 181-188 April-June 2012 


Peziza michelii and its ectomycorrhizae with 
Alnus nitida (Betulaceae) from Pakistan 


T. ASHRAF’, M. HANIF? & A. N. KHALID? 


Department of Botany, University of the Punjab, Quaid-e-Azam Campus, Lahore, 54590, Pakistan 
CORRESPONDENCE TO *: *tayibamaan01@gmail.com, 
*mhanif_r@hotmail.com, *drankhalid@gmail.com 


ABSTRACT — Peziza michelii and its ectomycorrhizae with Alnus nitida are characterized 
morphologically and molecularly by nrDNA ITS1-5.8S-ITS2 sequence analyses. Both the 
fungus and its morphotype are new records for Pakistan. 


Key worps — operculate asci, Pezizales, phylogeny 


Introduction 

Peziza Dill. ex Fr. (Pezizales, Pezizaceae) is a large, widely distributed 
heterogeneous genus represented by 104 species (Kirk et al. 2008). Its trophic 
status ranges from saprobic to mycorrhizal (Hansen et al. 2001). 

Identification of pezizalean mycorrhizae using morphological or 
conventional methods has remained difficult since they lack hyphal strands, 
connecting apothecium, and diagnostic morphotypes. Generally pezizalean 
ECM (ectomycorrhizae) possess a thin pseudoparenchymatous mantle, 
infrequent emanating hyphae, and rhizomorphs, and clamp connections are 
lacking (Agerer 1991, 2001). At the present time molecular tools are quite 
helpful for ECM identification, and the ECM of many Peziza species have been 
confirmed using such tools. Species identified as mycorrhizal with deciduous 
trees include Peziza depressa Pers., P. michelii, P. ostracoderma Korf, P. succosa 
Berk., and P. succosella (Le Gal & Romagn.) M.M. Moser ex Aviz.-Hersh. & 
Nemlich (Tedersoo et al. 2006). 

Betulaceae is represented in Pakistan by two genera, Alnus and Betula (Naisr 
1975), for which there are no previous reports about ectomycorrhizal status 
from Pakistan. Previously, P michelii has been reported as ectomycorrhizal 
with Alnus sp., Picea abies (Tedersoo et al. 2009), Betula sp. (Tedersoo et al. 


182 ... Ashraf, Hanif & Khalid 


2008), Fagus orientalis (Bahram et al. 2012) Tsuga canadensis (McLenon-Porter 
2008), and Tilia sp. (Lang et al. 2011). Mycorrhizal associations of Alnus with 
P. michelii are confirmed molecularly in this study. 

Ahmad et al. (1997) and Ashraf & Khalid (2012) have reported 89 
pezizalean taxa (including nine Peziza species) from Pakistan, and ascocarp 
surveys in Pakistan account for eight known to be ectomycorrhizal. Molecular 
characterization of Pezizales and their ectomycorrhizae in Pakistan is in 
progress. Our study, which documents association of P michelii with Alnus nitida 
(Spach) Endl. in Asia for the first time, is the first attempt at morphological and 
molecular characterization of ascomycete morphotypes from Pakistan. 


Materials & methods 

Ascomata were collected and photographed; necessary data was recorded in field 
and specimens were dried with the help of a fan heater. Ascomata were rehydrated and 
free hand sections were made in the laboratory. Microscopic (morpho-anatomical) 
characters were noted at 10x and 40x and drawings were made with the help of camera 
lucida. 

Soil under A. nitida was sampled and washed to isolate, characterize, and identify 
ECM. ‘The selected morphotypes were characterized morphologically following Agerer 
(2001) and have been deposited in Herbarium of Botany Department (LAH), University 
of the Punjab in Lahore. 

DNA was extracted from dried ascomata and morphotypes using Extract N. Amp. 
™ Plant kit (SIGMA). The nrDNA ITS1-5.8S-ITS2 region was amplified using fungal 
specific primers pairs (ITS1F/ITS4) following manufacturer's protocol with denaturation 
at 94°C for 4 min, followed by 35 cycles of 45 sec at 94°C, 45 sec at 54°C, and 1 min 
30 sec at 72°C, and a final extension at 72°C for 2 min. After purification, the PCR 
products underwent bidirectional sequencing by Macrogen (South Korea). GenBank 
ITS sequences were BLAST-searched for sequence comparison and identification. 
Selected sequences were aligned using Clustal W and corrected manually. All ambiguous 
insertions and deletions were removed prior to further analyses. Sequences have been 
accessioned in GenBank. 


Results 

Alnus nitida roots were found to be colonized by Peziza michelii. The BLAST 
search of the ITS sequences from P michelii ascomata and morphotypes 
associated with Alnus nitida obtained in the present study showed a 99% 
identity and 98% query coverage match with P michelii from Denmark 
(DQ200839.1). The identity percentage was calculated at 96.3, showing a 
97% identity and minimum divergence of 1.5 and 0.8 compared with Peziza 
michelii (DQ200838.1 and DQ200839.1) respectively (PLATE 1). The topology 
and identification of P. michelii was confirmed by the neighbor-joining method 


Peziza michelii +Alnus nitida ectomycorrhizae (Pakistan) ... 183 


and maximum likelihood method (PLaTEs 2-3). Both analyses place P. michelii 
with P. succosa and P. succosella as sister clade. 


Peziza_michelii_ENA38845_Pakistan 
Peziza_michelii_FR852088.1_Iran 
Peziza_michelii_DQ200839.1_Denmark 
Peziza_michelii_DQ200838.1_Denmark 
Peziza_infossa_DQ974817.1_USA 
Peziza_cf.__succosa_EU819417 
Peziza_succosella_DQ200841.1_Denmark 
Peziza_succosa_DQ200840.1_Denmark 
Peziza_phyllogena_AY789329.1_USA 
Peziza_ostracoderma_JN002180 
Chromelosporium_carneum_FJ7911 
Peziza_stuntzii_FJ268642.1_USA 
Cazia_flexiascus_EU846309 
Peziza_depressa_DQ200837 
Mycoclelandia_arenacea_Q231745 
Peziza_michelii_TA116_Pakistan 


Divergence 


Pate 1. Percent divergence and identity calculated by comparing sequence pairs reconstructed by 
MegAlign (DNASTAR). 


Cazia_flexiascus_EU846309 
Mycoclelandia_arenacea_Q231745 
Peziza_stuntzii_FJ268642.1_USA 
Peziza_depressa_DQ200837 
Peziza_ostracoderma_JN002180 
Peziza_phyllogena_AY789329.1_USA 
Chromelosporium_carneum_FJ7911 
Peziza_infossa_DQ974817.1_USA 
Peziza_cf.__succosa_EU819417 
Peziza_succosella_DQ200841.1_Denmark 
Peziza_succosa_DQ200840.1_Denmark 
Peziza_michelii_ENA38845_Pakistan 
Peziza_michelii_TA116_Pakistan 
Peziza_michelii_DQ200839.1_Denmark 
Peziza_michelii_DQ200838.1_Denmark 
Peziza_michelii_FR852088.1_Iran 
Sa ne er) Pee a | 


295, 9 20- 15° 10, = JPG 
Nucleotide Substitutions (x100) 


29.6 


PLATE 2. Phylogenetic tree based on ITS1-5.8S-ITS2 nucleotide sequences adjusted to 722 bases and 
constructed by the neighbor-joining method using MegAlign. The scale indicates the percentage 
of base difference (% divergence). Sequence data for phylogenetic analysis were taken from the 
GenBank nucleotide sequence database for Peziza, Cazia, Chromelosporium, and Mycoclelandia. 


184 ... Ashraf, Hanif & Khalid 


99 - Peziza_michelii_ENA38845_Pakistan 
50] L Peziza_michelii_TA116_Pakistan 
41 || Peziza_michelii_FR852088.1_Iran 
1001 Peziza_michelii_DQ200839.1_ Denmark 
100 Peziza_michelii_DQ200838.1_Denmark 
Peziza_succosella_DQ200841.1_ Denmark 
100 Peziza_succosa_DQ200840.1_Denmark 
53 Peziza_infossa_DQ974817.1_USA 
85 Peziza_cf._succosa_EU819417.1_ 
67 Peziza_phyllogena_AY 789329.1_USA 
47 Chromelosporium_cameum_FJ7911 
Peziza_ostracoderma_JN002180.1 
Mycoclelandia_arenacea_Q231745 
Cazia_flexiascus_EU846309.1 
40 Peziza_stuntzii_FJ268642.1_USA 
25 Peziza_depressa_DQ200837.1 
Cater o 


PiaTE 3. Molecular Phylogenetic analysis was inferred by using the Maximum Likelihood method. 
The tree with the highest log likelihood (-6466.0969) is shown. Bootstrap values are shown 
against each branch. Initial tree(s) for the heuristic search were obtained automatically: maximum 
parsimony method was used when common sites numbered <100 (or <1/4 total sites); otherwise 
BIONJ method with MCL distance matrix was used. The tree is drawn to scale, with branch lengths 
measured in number of substitutions per site. The analysis involved 16 sequences. 


Taxonomy 


Peziza michelii (Boud.) Dennis, Brit. Cup Fungi & Allies: 15 (1960) PLaTE 4 

APOTHECIA cupulate, medium-sized, diam. up to 5 cm, deeply concave, 
margins entire, but turning inwards, somewhat raised and torn, hymenium 
reddish brown with purplish tinge, smooth, outer surface yellow to yellowish 
brown, sessile to subsessile, solitary. HYMENIUM 290 um thick. Ascr cylindrical, 
operculate, unitunicate, 8-spored, uniseriate, strongly amyloid at apex, but 
diffusely blue along length, slightly narrowing near base, 260-278(-300) x 
13-17 um. AscosporEs uniseriate, ellipsoid, biguttulate, ornamented, 15-17(- 
19) x 9-10 um, ornamentation verrucose to warted: which appear elongated, 
irregularly and widely spaced, 0.3-0.7um high. PARApHysES long, slender, 
septate, with light brownish content, straight, 2-3 um wide, slightly thick up 
to 5-6 um at tip. ExcrpuLum: Ectal excipulum a textura globulosa to angularis, 
100-160 um thick, cells hyaline to slightly brown, thin-walled, diam. 10-22 


Peziza michelii +Alnus nitida ectomycorrhizae (Pakistan) ... 185 


PiatE 4. Peziza michelii: A. Apothecium. B. Apothecial section showing hymenium, 
sub-hymenium, medullary, and ectal excipulum. C. Part of hymenium, showing asci 
with ascospores and paraphyses. D. Ascospores. E. Excipulum. Scale bars: A = 0.5 cm; 
B = 90 um; C = 30 um; D =6 um; E = 80 um 


um; medullary excipulum a textura globulosa, 500 um thick, larger sub-globose 
thin-walled cells, 22-40 um diam., interspersed with numerous interwoven 
delicate hyphae <8 um diam. 


MATERIAL EXAMINED: PAKISTAN: KHYBER PAKHTUNKHWA, Khanspur, Helipad, 
Himalayan moist temperate forests, 2575 m (8205 ft) altitude, solitary, on ground, mossy 
substrate, 21 Aug. 2010, T. Ashraf TA-116 (LAH 210810; GenBank JN836748). 


Morphological description PLATE 5 
ECTOMYCORRHIZAL SYSTEM simple, axis 4-5 x 1.0-1.3 mm. UNRAMIFIED 
ENDS rounded to bent, bifurcate, 2-3 mm long and 0.8-1.2 mm in diam., 


186 ... Ashraf, Hanif & Khalid 


PLATE 5. ECM of Peziza michelii: A-B. Habit. C. Pseudoparenchymatous outer mantle. 
D. Pseudoparenchymatous inner mantle. E. Emanating hyphae. Scale bars: A=0.5 mm; 
B= 0.5 mm; C-D = 2 um; E= 1.5 um. 


younger tips creamy white, older tips brown to black. Texture of system felty to 
velvety with matte luster, host tissue not visible under the sheath. RHIZOMORPH 
absent. EMANATING HYPHAE rare, straight. 

MANTLE pseudoparenchymatous in all layers, OUTER MANTLE 
pseudoparenchymatous with irregular (ovoid to epidermoid) cells, hyphal cells 
infrequent and inconspicuous, hyphae without clamps, light yellowish, no cell 


Peziza michelii +Alnus nitida ectomycorrhizae (Pakistan) ... 187 


en 


PLATE 6. Cross section of Alnus nitida ectomycorrhizal root: m = mantle, ec = ectoderm, c = cortex, 
en = endoderm, cc = central cylinder, hn = hartig net. 


contents visible, cells 14-15 x 10-14um. INNER MANTLE pseudoparenchymatous, 
cells colorless to light yellowish, cells roundish to irregular in shape, no matrix 
material observed, cells 13-15 x 11-14 um. 

MANTLE THICKNESS 36 um, < 5 layers thick, discernible, cells rectangular 
to tangentially oval and/or tangentially elliptical. HARTIG NET paraepidermal, 
followed by large cortical cells, endodermis and central cylinder (PLATE 6) 

MATERIAL EXAMINED: PAKISTAN: KHYBER PAKHTUNKHWA, Khanspur, Helipad, 


Himalayan moist temperate forests, 2575 m (8205ft) altitude, roots of Alnus nitida, 
15 May 2010, M Hanif ENA38845 (LAH 150509; GenBank JN836749). 


Discussion 

The present work is the first report of Peziza michelii from Pakistan. This 
species can be found frequently in Himalayan moist temperate forests of 
Pakistan. Phylogenetically, P michelii clusters with the similar species from 
USA, Iran, and Denmark. 

Tedersoo et al. (2006) studied the morphotypes of P. michelii morphologically 
and used molecular markers to identify the mycobiont. They found these 
ectomycorrhizae to be solitary, rare, in small clusters, minute, conspicuous, and 
yellow green to olive green. This finding differed from the present report and 
morphotypes associated with Alnus nitida from Pakistan. Those morphotypes 
we studied are simple, creamy white with brown to black older tips, and lacking 
a rhizomorph. The anatomical features of the mantle surface also differ slightly 
in both morphotypes, with a mantle surface varying from smooth to finely 
granular in the morphotype reported by Tedersoo et al. (2006) versus the 
A. nitida morphotype, which is felty to velvety with matte luster with almost 
equal thickness (2-4 hyphal layers vs. 5 hyphal layers). Other mantle features in 
both studies also varied. The morpho-anatomic features of both morphotypes 


188 ... Ashraf, Hanif & Khalid 


of Peziza michelii may vary because of interaction of mycobiont with different 
photobionts. 


Acknowledgements 

The authors would like to thank Dr. Donald H Pfister (Harvard University Herbaria, 
Cambridge USA) and Dr. Mohammad Bahram (University of Tartu. Estonia) for their 
helpful comments and critical review of this manuscript. 


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Ahmad §S, Iqbal SH, Khalid AN. 1997. Fungi of Pakistan. Sultan Ahmad Mycological Society of 
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Hyrcanian forests of northern Iran. New Phytol. 193(2): 465-473. 
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Hansen K, Lzessge T, Pfister DH. 2001. Phylogenetics of the Pezizaceae, with an emphasis on Peziza. 
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Kirk PM., Cannon PF, David JC, Stalpers JA. 2008. Ainsworth and Bisby’s dictionary of the fungi, 
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Lang C, Seven J, Polle A. 2011. Host preferences and differential contributions of deciduous tree 
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McLenon-Porter TM. 2008. Above and below ground fungal diversity in a hemlock-dominated 
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Nasir YJ. 1975. Betulaceae. 1-5, in: E Nasir, SI Ali (eds). Flora of Pakistan. No. 95. Department of 
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Tedersoo L, Jairus T, Horton BM, Abarenkov K, Suvi T, Saar I, Kéljalg U. 2008. Strong host 
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Tedersoo L, Suvi T, Jairus T, Ostonen I, Pélme S. 2009. Revisiting ectomycorrhizal fungi of the 
genus Alnus: differential host specificity, diversity and determinants of the fungal community. 
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ISSN (print) 0093-4666 © 2012. Mycotaxon, Ltd. ISSN (online) 2154-8889 


MY COTAXON 


http://dx.doi.org/10.5248/120.189 
Volume 120, pp. 189-194 April-June 2012 


Erysiphe javanica sp. nov., 
a new tropical powdery mildew from Indonesia 


JAMJAN MEEBOON’, IMAN HIDAYAT? & SUSUMU TAKAMATSU’™* 


"Department of Bioresources, Graduate School, Mie University, Tsu 514-8507, Japan 

?Microbiology Division, Research Center for Biology, Indonesian Institute of Sciences, 
Cibinong 16911 West Java, Indonesia 

* CORRESPONDENCE TO: takamatu@bio.mie-u.ac.jp 


Axsstract — During the collection of tropical powdery mildews in Cibodas Botanical 
Garden and Mount Tangkuban Perahu in Indonesia, we found interesting specimens on 
Castanopsis javanica (Fagaceae). Phylogenetic analysis using a combination of 28S and ITS 
rDNA sequences clearly showed that this fungus forms a distinct lineage among the Erysiphe 
species found on hosts of the plant genus Castanopsis. Therefore, we consider this fungus a 
new species of Erysiphe, described here as Erysiphe javanica. Differences between E. javanica 
and closely related Erysiphe species are discussed. 


Key worps — Asia, Brasiliomyces, Erysiphaceae, phytopathogenic fungi, tropic 


Introduction 

Species of Erysiphe R. Hedw. ex DC. on fagaceous plants have been reported as 
a unique group of powdery mildews because they comprise not only fungi with 
simple mycelioid appendages that are characteristic for Erysiphe sect. Erysiphe 
(Braun & Takamatsu 2000), but also encompass taxa with different types of 
chasmothecial appendages previously belonging to the genera Brasiliomyces 
Viégas [rudimentary/mycelioid], Uncinula Lév. [uncinuloid], and Microsphaera 
Lév. [dichotomously branched at the apex] (Braun & Takamatsu 2000, 
Takamatsu et al. 2007, Divarangkoon et al. 2011). In addition, Divarangkoon 
et al. (2011) reported that the lineage of fagaceous Erysiphe spp. consists of 
not only taxa with multiple peridial cell layers but also species characterised 
by a single peridial cell layer previously assigned to Brasiliomyces [i.e., E. trina 
Harkn. (= Brasiliomyces trinus (Harkn.) R.Y. Zheng), E. asiatica Meeboon et al., 
and E. monoperidiata Meeboon et al. (Divarangkoon et al. 2011)]. These results 
suggest that different appendage types and single-layered peridial cells evolved 
on fagaceous hosts. 


190 ... Meeboon, Hidayat & Takamatsu 


Several authors (Zheng 1984, Braun 1987, To-anun et al. 2003) have discussed 
the significance of the peridial cells in separating Brasiliomyces from Erysiphe. 
Zheng (1984) noted that Brasiliomyces must be treated as an independent genus 
due to having a single layer of peridial cells not differentiated into inner and 
outer layers as described in the genus Cystotheca Berk. & M.A. Curtis. This 
argument was supported by Braun (1987) based on the reexamination of the 
type species, B. malvastri Viégas. Before 2011, eight Brasiliomyces species 
had been described based on this morphological criterion (To-anun et al. 
2003; Divarangkoon et al. 2011), of which there were recorded on fagaceous 
host plants — B. trinus (= E. trina), B. cyclobalanopsidis K.C. Kuo et al. 
(= E. cyclobalanopsidis (K.C. Kuo et al.) U. Braun), B. kumaonensis N. Ahmad et 
al. (= E. kumaonensis (N. Ahmad et al.) U. Braun). Braun & Paul (2009) placed 
these three species on fagaceous hosts in synonymy under Erysiphe, because 
E. trina nested in the lineage of Erysiphe emend. Braun & Takamatsu (2000). 

During a March 2011 visit to the Cibodas Botanical Garden and Mount 
Tangkuban Perahu on Java Island (Indonesia), we found specimens 
morphologically similar to E. asiatica and E. monoperidiata. Molecular analyses 
showed that the rDNA sequences of the specimens form an independent lineage 
separated from E. asiatica and E. monoperidiata sequences. We therefore 
propose this fungus as a new species. 


Materials & methods 


Morphological examination 

Specimens were collected in two locations of West Java province (Indonesia), 
namely, Cibodas Botanical Garden, Bogor and Mount Tangkuban Perahu, Bandung. 
All collections were conducted in March 2011. Details of host name, collection date, 
place, and collector were recorded. Morphological examinations were carried out as 
outlined in Divarangkoon et al. (2011). Mycelia and chasmothecia were stripped from 
leaf surfaces with a clean needle, mounted on a microscope slide, and examined in 
3% NaOH using a light microscope with phase contrast 10x, 20x, and 40x objectives. 
Thirty chasmothecia, asci, and ascospores were measured per sample. Specimens were 
deposited at the National Museum of Nature and Science (TNS), Japan; Mie University 
Mycological Herbarium (MUMH), Japan; and Herbarium Bogoriense (BO), Indonesia. 


Phylogenetic analysis 

Whole-cell DNA was extracted from chasmothecia using the chelex method (Walsh 
et al. 1991) as described in Hirata & Takamatsu (1996). The 5’-end of the 28S rDNA, 
including the domains D1 and D2, and ITS region, including the 5.88 rDNA, were 
amplified by polymerase chain reaction (PCR) and then sequenced using protocols 
as described in Takamatsu et al. (2006). Representative sequences determined in this 
study were deposited in DNA databases (DDBJ, EMBL, GenBank) under the accession 
numbers of JQ220151-JQ220162. Sequences generated from the rDNA ITS region 
and D1/D2 domains were aligned using MEGA 5 (Kumar et al. 2008) with Erysiphe 


Erysiphe javanica sp. nov. (Indonesia) ... 191 


and Brasiliomyces sequences retrieved from DNA databases (DDBJ, EMBL, GenBank). 
The alignment was deposited in TreeBASE (http://www.treebase.org/) under the 
accession number of $12121. Maximum parsimony (MP) analysis was done in PAUP* 
4.0b10 (Swofford 2002) with the heuristic search option using the ‘tree-bisection- 
reconstruction (TBR) algorithm. All sites were treated as unordered and unweighted, 
with gaps treated as missing data. The strength of the internal branches of the resulting 
tree was tested with bootstrap analysis using 1000 replications (Felsenstein 1985). Tree 
scores, including tree length, CI, RI, and RC, were also calculated. 


Taxonomy 


Erysiphe javanica Meeboon & S. Takam., sp. nov. FIG. 1 
MycoBank MB 563656 


Similar to Erysiphe asiatica, but differing in 5-7-spored asci and occurrence on 
Castanopsis javanica. 


TYPE: on Castanopsis javanica (Blume) A. DC. (Fagaceae), Indonesia, West Java 
province, Bogor, Cibodas Botanical Garden, 14 March 2011, J. Meeboon, I. Hidayat & 
S. Takamatsu (Holotype: TNS-F-44236; isotype: MUMH 5153; ex-type rDNA sequences, 
JQ220160 (28S), JQ220162 (ITS)). 


Erymo.ocy: the species epithet refers to the island where the specimens were 
collected. 
Cotonies hypophyllous, persistent, forming irregular white patches on the 
host surfaces. HypHAE hyaline, superficial, septate, branched, 3-6 um wide. 
APPRESSORIA well-developed, coral-like, single or occasionally opposite in 
pairs. CONIDIOPHORES and CONIDIA unknown. CHASMOTHECIA scattered to 


Fic. 1. Erysiphe javanica sp. nov.: 1-3, chasmothecia; 4—6, asci with 5—7 spores. Bars = 50 um. 


192 ... Meeboon, Hidayat & Takamatsu 


gregarious, (41-)50-66(-69) um diam., globose, containing 2 asci; appendages 

present, rarely absent, poorly developed, mycelioid, (8—)17-73(-95) x (2-)3-5 

(-8), colourless, aseptate, thin-walled, smooth. PERtpIuM thin, one conspicuous 

layer, yellowish to light brown, semitransparent. Asci globose to subglobose, 

sessile or short-stalked, (30-)36-50(-76) x (26-)27-44(-53) um, 5-7-spored. 

Ascosporss ellipsoid-ovoid, hyaline, (10-)11-24(-26) x (5-)8-14(-17) um. 
ECOLOGY & DISTRIBUTION: on Castanopsis javanica, Indonesia. 


ADDITIONAL COLLECTIONS EXAMINED: on Castanopsis javanica: INDONESIA. WEsT 
JAVA PROVINCE, Bogor, Cibodas Botanical Garden, 14 March 2011, J. Meeboon, I. 
Hidayat & S. Takamatsu (MUMH 5147, BO22660, BO22661, BO22662, BO22663); 
Bandung, Mount Tangkuban Perahu, 12 March 2011, J. Meeboon, I. Hidayat & 
S. Takamatsu (MUMH 5123, BO22655, BO22656, BO22657, BO22658, BO22659). 


Phylogenetic analysis 

In the 28S+ITS combination sequence data set, 1091 of 1277 total characters 
used in the MP analysis were constant, 58 characters were variable and 
parsimony-uninformative, and 128 characters were parsimony-informative. 
A total of 240 equally parsimonious trees (TL = 212, CI = 0.934, RI = 0.924, 
RC = 0.863) were generated by the MP analysis (Fic. 2). All Erysiphe species 
found on Castanopsis or Lithocarpus form a monophyletic clade with 98% 
bootstrap support. Erysiphe javanica, E. asiatica, and E. monoperidiata form 
independent clades with 99%, 100% and 66% bootstrap supports, respectively. 


69| Erysiphe monoperidiata MUMH4990 

53 Erysiphe monoperidiata MUMH4991 
Erysiphe monoperidiata MUMH4988 

66l| Erysiphe monoperidiata MUMH4985 
Erysiphe monoperidiata MUMH4986 
Erysiphe monoperidiata MUMH4987 

99| Erysiphe javanica MUMH5147 
Erysiphe javanica MUMH5153 

100 | Erysiphe asiatica MHMU4989 

Erysiphe asiatica MUMH 4992 

Erysiphe gracilis ex Quercus glauca AB022358 
Erysiphe japonica ex Quercus AB022416 


Erysiphe trina ex Quercus AB022351 


100| Erysiphe necator ex Vitis DQ444327 


Erysiphe necator ex Vitis DQ189089 


~~~ 5 changes 


Fic. 2. Phylogeny of Erysiphe javanica inferred from a combination of 28S + ITS rDNA sequences 
using maximum parsimony (MP) method. Bootstrap support percentages (1000 replications; 
>50%) are shown on the branches. 


Erysiphe javanica sp. nov. (Indonesia) ... 193 


TABLE 1. Morphological comparison of Erysiphe javanica, E. monoperidiata, 
and E. asiatica. 


CHARACTER E. javanica E. monoperidiata E. asiatica 

COLONIES Hypophyllous Amphigenous, Hypophyllous 
mainly epiphyllous 

HYPHAE 3-6 4-6 4-6 


(um diameter) 


CHASMOTHECIA 
(um diameter) 


(41-)50-66.5(-69.5) 
2 asci 


(55.5-)58-82.5(-85) 
2-4 asci 


(51-)57-74(-78) 
2 asci 


APPENDAGE (8.5-)17.5-73.5(-95) x (15.5-)18-66(-75) x (31-)45-51(-66) x 
(um) (2-)3.5-5.5(-8) (2.5-)3-6(-7.5) (4-)4.5-5(-5.5) 
ASCI (30.5-)36—50.5(-76) x (34-)36-58(-61) x (45-)46-59(-62) x 

(um) (26-)27.5-44(-53) (24-)28-49(-52) (38-)40-53(-57.5) 
5—7-spored 4—6-spored 6—8-spored 
ASCOSPORES (10.5-)11.5-24.1(-26) x (11-)12.5-25(-26) x (16-)18-25(-28) x 
(um) (5-)8-14(-17) (6-)7.5-13(-14.5) (8.5-)9-15(-16.5) 
Discussion 


Among the seven Erysiphe species previously found on Castanopsis (Braun 
1987; Divarangkoon et al. 2011), only E. asiatica and E. monoperidiata were 
reported to have a chasmothecial peridium composed of a single cell layer 
(Divarangkoon etal. 2011). Morphologically, there are only slight morphological 
differences between E. javanica, E. asiatica, and E. monoperidiata (TABLE 
1). Erysiphe javanica differs from E. monoperidiata in having hypophyllous 
colonies, smaller chasmothecia (69 um diam. in E. monoperidiata), having only 
2 asci (with 5-7 spores per ascus) per chasmothecium, and being found only 
on C. javanica in Indonesia. In addition, the morphologically close E. asiatica 
differs slightly in having 6-8 spored asci and being confined to C. diversifolia and 
C. echinocarpa in Thailand. Although it is difficult to differentiate E. javanica 
from E. asiatica and E. monoperidiata solely on morphology, our phylogenetic 
analysis, using a combination of 28S and ITS rDNA sequences, clearly showed 
that E. javanica forms a distinct lineage distinguishable from E. asiatica and 
E. monoperidiata (Fic. 2). 


Acknowledgments 

This work was financially supported in part by a Grant-in-Aid for Scientific 
Research (No. 23580061) from the Japan Society of the Promotion of Science to ST 
and MONBUKAGAKUSHO: MEXT (Ministry of Education, Culture, Science, and 
Technology) Scholarship of the Japanese Government awarded to JM. The authors 
also thank the Research Center for Biology, Indonesian Institute of Sciences (LIPI) for 
providing facilities during collection and examination of powdery mildew samples. 


194 ... Meeboon, Hidayat & Takamatsu 


Literature cited 

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Braun U, Paul YS. 2009. The Indian Erysiphaceae revised. Nova Hedwigia 89: 371-395. 

Braun U, Takamatsu S. 2000. Phylogeny of Erysiphe, Microsphaera, Uncinula (Erysipheae) and 
Cystotheca, Podosphaera, Sphaerotheca (Cystotheceae) inferred from rDNA ITS sequences— 
some taxonomic consequences. Schlechtendalia 4: 1-33. 

Divarangkoon R, Meeboon J, Monkhung S, To-anun C, Takamatsu S. 2011. Two new species of 
Erysiphe (Erysiphales, Ascomycota) from Thailand. Mycosphere 2: 231-238. 

Felsenstein J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 
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http://dx.doi.org/10.1007/BF02461299 

Kumar S, Nei M, Dudley J, Tamura K. 2008. MEGA: A biologist-centric software for 
evolutionary analysis of DNA and protein sequences. Briefings in Bioinformatics 9: 299-306. 
http://dx.doi.org/10.1093/bib/bbn017 

Swofford DL. 2002. PAUP*: Phylogenetic Analysis Using Parsimony (*and Other Methods). 
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Takamatsu S, Matsuda S, Niinomi S, Havrylenko M. 2006. Molecular phylogeny supports a Northern 
Hemisphere origin of Golovinomyces (Ascomycota: Erysiphales). Mycological Research 110: 
1093-1101. http://dx.doi.org/10.1016/j.mycres.2006.07.005 

Takamatsu S, Braun U, Limkaisang S$, Kom-un S, Sato Y, Cunnington JH. 2007. Phylogeny and 
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To-anun C, Limkaisang S, Fangfuk W, Sato Y, Braun U, Takamatsu S. 2003. A new species of 
Brasiliomyces (Erysiphaceae) on Dalbergia cultrata var. cultrata from Thailand. Mycoscience 44: 
447-451. http://dx.doi.org/10.1007/s10267-003-0140-1 

Walsh SP, Metzger DA, Higuchi R. 1991. Chelex 100 as a medium for simple extraction of DNA for 
PCR-based typing from forensic material. Biotechniques 10: 506-513. 

Zheng RY. 1984. The genus Brasiliomyces (Erysiphaceae). Mycotaxon 19: 281-289. 


ISSN (print) 0093-4666 © 2012. Mycotaxon, Ltd. ISSN (online) 2154-8889 


MY COTAXON 


http://dx.doi.org/10.5248/120.195 
Volume 120, pp. 195-208 April-June 2012 


Pisolithus: a new species from southeast Asia 
and a new combination 


C. PHosri?’, M.P. MARTIiN?, N. SUWANNASAL, P. SIHANONTH* & R. WATLING? 


"Microbiology programme, Pibulsongkram Rajabhat University, Phitsanulok, 65000, Thailand 
*Departamento de Micologia, Real Jardin Botanico, CSIC, 

Plaza de Murillo 2, 28014, Madrid, Spain 
*Department of Biology, Srinakharinwirot University, Bangkok, 10110, Thailand 
‘Department of Microbiology, Faculty of Science, Chulalongkorn University, 

Bangkok, 10330, Thailand 
°Caledonian Mycological Enterprises, 

Crelah, 26 Blinkbonny Avenue, Edinburgh, EH4 3HU, Scotland 
*CORRESPONDENCE TO: cherd_phosri@yahoo.co.uk 


ABSTRACT — Based on morphological and ITS nrDNA sequence analyses, a species 
recognized in classical European literature as Lycoperdon capsulifer is transferred to Pisolithus 
and P. orientalis is proposed as a new species. Pisolithus orientalis basidiomes, collected under 
Pinus kesiya in Thailand, correspond to Pisolithus sp. 5 sensu Martin et al. Pisolithus sp. 4 
sensu Martin et al. is shown to comprise two groups, one equivalent to P. arhizus as collected 
from Pinus and Quercus forests from Italy and Spain and the other represented by the new 
P. capsulifer from mixed pine-deciduous forests in England and France. 


KEY worps — ectomycorrhizal fungi, gasteromycete, phylogenetic species, PT, taxonomy 


Introduction 

Pisolithus is one of the most widespread and cosmopolitan gasteromycete 
genera ranging from temperate to tropical regions of the world. It has long 
been considered as monotypic (Coker & Couch 1928, Cunningham 1942, 
Pilat 1958) and is well-researched, primarily based on ectomycorrhizal 
studies, as Pisolithus tinctorius (P. Micheli ex Pers.) Coker & Couch (Marx et 
al. 1984, Burgess et al. 1995). Rauschert (1959) proposed that this species was 
conspecific with Polysaccum arhizum Scop., which he recombined as Pisolithus 
arhizus (Scop.) Rauschert. However, after molecular studies indicated that 
P. tinctorius is a species complex (Anderson et al. 1998, Cairney et al. 1999, 
Diez et al. 2001), Martin et al. (2002) proposed at least 11 phylogenetic species. 
Several subsequent morphological and molecular studies have confirmed 


196 ... Phosri & al. 


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Pisolithus orientalis sp. nov. (Thailand) ... 197 


the taxonomic rank of some old species [Pisolithus albus (Cooke & Massee) 
Priest, P marmoratus (Berk.) E. Fisch., PB microcarpus (Cooke & Massee) 
G. Cunn.] and established several new ones (Pisolithus abditus Kanch. et al., 
P. aurantioscabrosus Watling, P. indicus Natarajan & Senthil.). Nonetheless, 
some clades recognised by Martin et al. (2002) remain without specific names. 

Additional morphological studies and ITS nrDNA sequence analyses of 
recently collected austro-asiatic, euro-asiatic, and paleotropical Pisolithus 
specimens have been conducted to assign morphological species to the 
Pisolithus sp. 5 phylogenetic lineage and to members of the AII clade of Martin 
et al. (2002). 


Materials & methods 


Fungal isolates 

The Pisolithus material used in this work included fresh, herbarium, or cultured 
specimens sampled from a variety of geographical sites (TABLE 1). Basidiomes for 
each isolate are retained in the herbarium at the Microbiology Programme, Faculty of 
Science & Technology, Pibulsongkram Rajabhat University and BBH Thailand. Cultures 
obtained from basidiomata were grown and maintained on Modified Melin Norkrans’ 
(MMN) medium (Marx & Kenny 1982) at 30°C with subculturing every 1-2 months. 
Herbarium specimens were provided by BCN and E (Abbreviations for herbaria follow 
Lanjouw & Stafleu 1964). 


Molecular methods 

Isolation, amplification, purification, and sequencing of DNA followed Phosri et al. 
(2007). DNA was isolated using the E.Z.N.A. fungi DNA miniprep kit (Omega Biotech, 
Doraville, Georgia, USA) as described by Martin & Garcia-Figueres (1999) or a DNeasy 
Plant Mini Kit (Qiagen, Hilden, Germany). ITS regions were amplified with Ready-To- 
Go™ PCR Beads (Amersham-Pharmacia Biotech, NJ, USA) (Martin & Winka 2000). 
Primer pairs ITS1F/ITS4B and ITS1/ITS4 were used to amplify the ITS1+5.8S+ITS2 
nrDNA gene cluster following White et al. (1990) and Gardes & Bruns (1993). Prior to 
sequencing, products were cleaned using either E.Z.N.A. Clean kit (Omega Biotech) or 
directly from the gel using QIAQuick PCR purification kit (QIAGEN, Chatsworth, CA). 
Both strands were sequenced separately at Secugen S.L. (Madrid, Spain) or Macrogen 
(South Korea). All samples were sequenced in both directions. 

When needed, DNA was cloned using pGEM®-T Easy Vector System II cloning kit 
(Promega Corporation, Madison, Wisconsin, USA) and purified with QIAPrep Spin 
Mini prep (QIAGEN). Both strands were sequenced separately using vector specific 
primers T7 and SP6 (Nag et al. 1988) at Secugen S.L. or Macrogen. 


Sequence analysis 

Navigator™ Sequence comparison software (Perkin Elmer Applied Biosystem) or 
Sequencher™ version 4.2 (Gene Codes Corporations, Ann Arbor, Michigan, USA) was 
used to assemble the consensus sequence from the two strands of the ITS nrDNA of 
each isolate. Blastn searches with megablast option were used to compare the sequences 
obtained against the sequences in the National Center of Biotechnology Information 


198 ... Phosri & al. 


(NCBI) nucleotide database. The new consensus sequences have been lodged in the 
EMLB-EBI database with accession numbers indicated in TABLE 1. 

Sequences obtained were compared with homologous sequences of Pisolithus spp. 
retrieved from the EMBL Nucleotide Sequence Database with many published in Martin 
et al. (2002), Kanchanaprayudh et al. (2003a,b), Moyersoen et al. (2003), and Reddy et 
al. (2005) to represent all their described clades. Prior to 2009 sequences were aligned 
using the SEQAPP software (Perkin Elmer Applied Biosystem) and thereafter using Se- 
Al v2.0a11 Carbon (Rambaut 2002). The alignment was optimized visually. Alignment 
gaps were indicated as “-” and ambiguous nucleotides were marked as “N”. 
Phylogenetic analyses 

The alignment was analysed using the programme PAUP 4.0b10 (Swofford 2003) and 
MrBAYES v3.0 b4 (Huelsenbeck & Ronquist 2001) as described in Telleria et al. (2010). 
Scleroderma citrinum Pers. (FM213344) and Suillus luteus (L.) Roussel (GU373495) 
were included as outgroups. First, a maximum parsimony analysis (MP) was inferred 
using the heuristic search option in PAUP. Gaps were treated as missing data. Branch 
lengths equal to zero were collapsed to polytomies. Nonparametric bootstrap (BP) 
support (Felsenstein 1985) for each clade was tested based on 10000 replicates, using 
the fast-step option. 

A second analysis was conducted using a Bayesian approach (Larget & Simon 1999, 
Huelsenbeck & Ronquist 2001). It was performed assuming the HKY + G model as 
suggested by hierarchical likelihood ratio test (hKRTs) and akaike information criterion 
(AIC) in MrModeltest 2.3 (Posada & Crandall 1998, Nylander 2004). 

A combination of both posterior probabilities and bootstrap proportion was used 
to assess the level of confidence for a specific node (Lutzoni et al. 2004). The alignment 
matrix and the 50% majority—rule consensus tree from the Bayesian analysis are available 
in TreeBase (http://www.treebase.org/). 

Recovered lineages and clades are named according to Martin et al. (2002). When a 
specific name has been formally assigned to a clade, the name is included along with the 
species number of Martin et al. (2002). 


Results 

Thirty-three new Pisolithus sequences have been generated, including those 
obtained from the epitype (E00290168) of the new combination, P. capsulifer, 
and the holotype (BBH28597) of the new species, P orientalis. These were 
aligned with 67 additional sequences obtained from databases (GenBank and 
UNITE; see Fics 1-2) and phylogenetically analyzed. 

The ITS nrDNA dataset contains 97 sequences and 893 aligned positions, of 
which 410 were constant, 177 parsimony uninformative, and 306 parsimony- 
informative. Maximum parsimony analysis yielded 100 most parsimonious 
trees (940 steps long, CI = 0.6681, HI = 0.3319, RI = 0.8968). One best tree 
found by branch swapping is shown in Fic. 1, with bootstrap values on the 
branches. The 50% majority-rule consensus tree from the Bayesian analyses is 
in Fic. 2, with posterior probabilities indicated on the branches. 


Pisolithus orientalis sp. nov. (Thailand) ... 199 


Suillus luteus, GU373495 
Scleroderma citrinum, ape 3344 F eaance ee fare Ralawsi 
, Shorea macroptera, Malaysia A : 
c AF415226, Shorea macroptera, Malaysia I P. aurantioscabrosus (P. species 11) 
AY756113, Vateria indica, India, === P. indicus (P. species 13) 
100 + AFO03915, Afzelia sp., Kenya ; 
AF228653, Afzelia sp., Kenya | P. species 1 
24PISOLI, Shorea sp., Thailand 
A I AB099921, Shorea roxburgii, Thailand J ‘ 
100 |- AB099920, Shorea roxburgii, Thailand P. abditus (P. species 12) 
31PISOLI, Shorea roxburgii, Thailand 
AB099922, Dipterocarpus alatus, Thailand 
AF003914, Eucalyptus camaldulensis, Kenya 
AF374/19, Eucalyptus caiopnylia Australia» 
, Eucalyptus calophylla, Australia f 
99 IF AF004734, Eucalyptus sp., New South Wales P. marmoratus (P. species 2) 
AF440866, Eucalyptus plantation, China 
AF374665, auc nes patens, Australia 
84 AF228641, Cistus ladanifer, Spain 
ARHPIS3 (clones a-c), Cistus ladanifer, Spain . 
400|| AF228642, Cistus ladanifer, Spain P. species 3 
AF228643, Cistus ladanifer, Spain 
AF228644, Cistus ladanifer, Spain ; 
AF374624, Acacia sp., Australia —=—_—=_< P. species 10 
AF347700, FUCAypUS, Brazil 
AF004735, Eucalyptus sp., New South Wales 
eee Busses she eee P. microcarpus 
, Eucalyptus sp., Brazi : 
95|L AF 140547, Eucalyptus sp.. Brazil (P. species 9) 
64 AF374704, Eucalyptus dunni, Brazil 
57 88 U62666, Eucalyptus citriodora, Brazil 
AF374661, Eucalyptus camaldulensis, Australia | P ies'S 
AF374708, Eucalyptus sp./Acacia sp., Australia 7SPECles 
AF004732, Eucalyptus sp., New South Wales =<  Pisolithus sp. 
AF270786, Eucalyptus sp., Australia 
AF374622, Acacia holosericea, Senegal 
72\I— .13PISOLI, Eucalyptus camaldulensis, Thailand 
14PISOLI, Eucalyptus camaldulensis, Thailand 
86 15PISOLI, Eucalyptus camaldulensis, Thailand 
AF228656, ueaipius sp., Spain 
AF228655, Eucalyptus sp., Morocco 
AF 228654, Eucalyptus sp., Morocco 


B ABO099918, Thailand 
99 AB099909, Eucalyptus camaldulensis, Thailand 
AF416589, Eucalyptus tereticornis, India P. albus complex 
AF440868, Eucalyptus sp., China (P. species 7) 


AF374675, Eucalyptus sp., Queensland 
AF004736, unknown potential host, New South Wales 
AF004737, unknown potential host, New South Wales 
17PISOLI, unknown potential host, Thailand 
AJ629887 (12PISOLI), Eucalyptus camaldulensis, Thailand 
20PISOLI, Eucalyptus sp., Hawaii, USA 
35PISOLI, Melaleuca sp., Thailand 
AB099911, Thailand 
AF374638, Acacia mangium, Malaysia 
21PISOLI, Eucalyptus Sp Australia ? 
AF374646, Eucalyptus globulus, Australia === P. species 10 
AF440867, Eucalyptus plantation, China === _- P. microcarpus 
AF003916, Pinus caribaea, pale 
AF228645, Quercus ilex/Q.coccifera, Spain 
AF228646, Quercus ilex/C. ladanifer, Spain ; 5 
72 posclls pins sp., sey P. tinctorius 
, Pinus sp., i 
AF 143234, unknown potential host, France (P. species 6) 
19PISOLI, Pinus sp./Quercus sp., USA 
AF 228647, Pinus caribaea, Kenya 
AF374625, Pinus kesiya, Thailand 
AF374679, Eucalyptus sp., China 
AF374711, Pinus sp./Eucalyptus sp., China 
AB099919, unknown potential host, Thailand 
53 34PISOLI, Pinus kesiya, Thailand K . 
pms enknown potent host Thailand P. orientalis 
LI, Pinus kesiya, Thailan - 
14PISOLI, Pinus kesiya, Thailand (P. species 5) 
10PISOLI, Pinus kesiya, Thailand 
33PISOLI, Pinus kesiya, Thailand 
All 7PISOLI, Pinus kesiya, Thailand 
8PISOLI, Pinus kesiya, Thailand 
FM213365 (3PISOLI), Pinus sp., Spain 
AF228648, Quercus ilex/Q. coccifera, Spain 
92bisPISOLI (clone c), Will Wood, Sardinia, Italy 
92bisPISOLI Go a, d), Will Wood, Sardinia, Italy 


92bisPISOLI (clone f), Will Wood, Sardinia, Italy ; 
AF228649, Q. ilex, Spain P. arhizus 
AF228651, Q. ilex, Spain (P. species 4) 


AF 228650, P. halepensis/Q. coccifera, Spain 
54 AF 228652, P. halepensis, Spain 
92bisPISOLI (clone b), Will Wood, Sardinia, Italy 
6PISOLI, Pinus sp., Spain 
AF374627, Pinus sp., South Africa ‘ 
AF374629, Pinus pumila/Betula ermanii, Japan 
77 UDB001206, Pinus sp. forest, Sweden 


51PISOLI, unknown potential host, England P. capsulifer 
46PISOLI, unknown potential host, England F 
954 S3PISOLI, mixed forest/Pinus sp., pranes (P. species 14) 


49PISOLI, Pinus sylvestris/P. contorta, England 


— 5changes 


Fic. 1. Phylogenetic tree (one of 100 most parsimonious trees) obtained with a parsimony analysis 
under heuristic search of ITS sequences of Pisolithus collections included in TABLE 1 and sequences 
obtained from the GenBank and UNITE. Each branch is labeled with the DNA isolation code in 
bold (new sequences) or with the accession number including the possible host and the country. 
Numbers above branches represent bootstrap values. Clades and species number according to 
previous authors. 


200 ... Phosri & al. 


In both analyses, Pisolithus sequences form a highly supported clade (BS = 
84%, PP = 1.0) with at least 15 terminal assemblages. However, the relationships 
between the clades are generally not well resolved and the main differences 
between Fic. 1 and Fie. 2 are related to the position of clades from lineage AI 
in Martin et al. (2002). 

The basal clade (BS <50%, PP = 0.62) (lineage C, Martin et al. 2002) included 
in our study contains Pisolithus aurantioscabrosus and P. indicus, which are 
sister to the remaining Pisolithus taxa. These two taxa are restricted to native 
dipterocarp forests in southeast Asia and India, respectively. 

Lineage AI is separated into four clades, each with high support (BP = 99% 
or PP = 1.0). Lineage AII also ramifies into at least four main groups, but these 
possess weak support (BS < 50%, PP = 0.98). The four AII groups are Pisolithus 
spp. 4, 5, and 6 of Martin et al. (2002), and one group here called Pisolithus sp. 
14. Pisolithus sp. 6 (BS = 72%, PP = 0.99) consisted of eight Pisolithus sequences 
from basidiomata found mainly in association with pine (Pinus caribaea 
Morelet.) and oak (Quercus ilex L., Q. coccifera L.) in various regions ranging 
from USA, Spain, Kenya, and France. We consider this clade to be P. tinctorius 
s. St., sister to the Pisolithus sp. 5 clade formed by seven new isolates associated 
with Pinus kesiya Royle ex Gordon in Thailand and five GenBank sequences. 
The unifying features unique to these collections lead us to describe them below 
as a new species, Pisolithus orientalis. The other two clades (Pisolithus spp. 4 
and 14) in Lineage AII are sister to the clade formed by Pisolithus tinctorius 
s. st. and P orientalis. Collections from the British Isles (46PISOLI, 49PISOLI 
and 51pisoL1) and France (53pisoLi) clearly separate from Pisolithus sp. 4 
(BS = 70%, PP = 1.0) and group together with sequences from Sweden 
(UDB001206) and Japan (AF374629). Sequenced specimens from British Isles, 
France, and Sweden have been analysed in parallel to basidiospore SEM studies. 
The electron micrographs (Fic. 3) indicate that the spore ornamentation does 
not resemble that of any other Pisolithus documented. The specific name 
Pisolithus capsulifer has been assigned to Pisolithus sp. 14. 


Discussion 

In lineage AII, one clade (‘sp. 14’) comprises predominantly British material. 
Pisolithus is very rare in the British Isles, where it is recorded from London, 
Hampshire, Devon, and Norfolk. Most records are from Tertiary gravels and 
sandy mineral soils and confined to the southernmost part, especially the 
southeast of England. There is one record of Pisolithus from Ireland ‘on a bank 
in a car park, under Castanea sativa and Pinus spp., well-drained peat over lying 
sandy boulder-clay north slope of Knockmealdon Mountains, Kilballyboy Wd., 
South Tipperary (July 1984)’ (Ing 1995), but it is not recorded from Scotland. 
The British sequences agreed with one each from France and Sweden. Here spore 
morphology is also distinctive, but is it significant? Grand (1976) and Watling 


Pisolithus orientalis sp. nov. (Thailand) ... 201 


Suillus luteus, GU373495 


Scleroderma citrinum, FM213344 pale: F 
AY756113, Vateria indica, Indica =—=_ +P. indicus (P. species 13) 


00 AF41 227, Shores macroptera, Malaysia } | P. aurantioscabrosus 
, Shorea macroptera, Malaysia i 
Al 4.00 - AF003915, Afzelia sp. Kenya % a (P. species 11) 


AF228683, Afzelia sp. Kenya_ | P. species 1 
A l AB099922, Dipterocarpus alatus, Thailand 
24PISOLI, Shorea sp., Thailand 5 F 
1.00 |} 31PISOLI, Shorea roxburgii, Thailand P. abditus (P. species 12) 
AB099920, Shorea roxburgii, Thailand 
AB099921 Shorea roxburgii, Thailand 
AF228641, Cistus ladanifer, Spain. . 
1.00 1.00 AF 228644, Cistus ladanifer, Spain ; 
ARHPIS3, (clones a-c), Cistus ladanifer, Spain P. species 3 
Al AF228643, Cistus ladanifer, Spain 
AF228642, Cistus ladanifer, Spain. 
0.88 4.00 AF374665, Eucalyptus patens, Australia 
: AF003914, Eucalyptus camaldulensis, Kenya 
‘AF374719, Eucalyptus caiophylla, Australia» P. marmoratus 
AF440866, Eucalyptus plantation, China (P. species 12) 
AF004734, Eucalyptus sp., New South Wales 
AF004733, Eucalyptus sp., New South Wales ? 
AF374646, Eucalyptus globulus, Australia P. species 10 
0.96 AF440867 Eucalyptus plantation China P. microcarpu: 
cacia sp., Australia 2. specie: ket 
B AF374624, Acaci Austral P. 
1.00 004732, Eucalyptus sp., New South Wales —_—_—=- Pisolithus sp. 
8210.99 , Eucalyptus camaldulensis, Australia F 
0.82 AF374661, Eucal /dulensis, Australia | 
AF374708, Eucalyptus sp./Acacia sp., Australia P. species 8 
0.99 AF374704, Eucalyptus dunni, Brasil 
: 9.99] 4 oof + U62666, Eucalyptus citriodora, Brasil 
AF 140547, Eucalyptus sp., Brasil. P. microcarpus 
AF347700, Eucal lyptus sp., Brasil J 
AF228657, Eucalyptus sp., Morocco (P. species 9) 
1.00 AF 142991, Eucalyptus sp., Brasil 
: AF004735, Eucalyptus sp., New South Wales 
AB099911, Thailand é 
20PISOLI, Eucalyptus sp.,. Hawaii, US 
0.80/L 35PISOLI, Melaleuca sp., Thailand 
AF374638, Acacia mangium, Australia_ 
17PISOLI, unknown potential host, Thailand __ : 
AJ629887 (PISOLI12), Eucalyptus camaldulensis, Thailand 
0.78 AF004737, unknown potential host, New South Wales 
i AF004736, unknown potential host, New South Wales 
21PISOLI, Eucalyptus sp., Australia 
AF440868, Eucalyptus sp., China 
AF374675, Eucalyptus 8p. Morocco _ “ P. albus complex 
AB099909, Eucalyptus camaidulensis, Thailand (P. species 7) 
0.99 AF416589, Eucalyptus tereticornis, India 
AF270786, Eucalyptus sp., Australia 
1.00 AF374622, Acacia holosericia, Senegal 
AB099918, Thailand : 
AF 228656, Eucalyptus sp., Spain 
AF228655, Eucalyptus sp., Morocco 
AF228654, Eucalyptus sp., Morocco _ , 
13PISOLI, Eucalyptus camaldulensis, Thailand 
15PISOLI, Eucalyptus camaldulensis, Thailand 
; ; 14PISOLI, Eucalyptus camaldulensis, Thailand 
1.00 ARS TAGES. Pinus pumila/Betula ermanii, Japan 


01206, Pinus sp. forest, Sweden - 
0.91 4 Soe mown potential host, En fang eae P. capsulifer 
; , Pinus sylvestris/P. contorta, Englan . 
0.56 46PISOLI, unkncwn potential host, England (P. species 14) 


L 53PISOLI, mixed forest/ Pinus sp., France 
AF374627, Pinus SP South Africa 
FM213365 (3PISOLI), Pinus sp., Spain 
1.04 6PISOLI, Pinus sp, Spain ie i 
92bisPISOLI (clone Wil Wood, Sardinia, Italia 
92bisPISOLI (clone b), Will Wood, Sardinia, Italia P. arhizus 
92bisPISOLI (clone c), Will Wood, Sardinia, Italia : 7 
92bisPISOLI (clone a), Will Wood, Sardinia, Italia (P. species 4) 
AF228648, Quercus ilex/Q. coccifera, Spain 
All AF228650, Pinus halepensis/Q.coccifera, Spain 
0.98 AF 228652, Pinus halepensis, Spain 
, AF228649, Quercus ilex, Spain 
AF228651, Quercus ilex, Spain 
AF003916, Pinus caribaea, Kenya 
19PISOLI, Pinus sp./Quercus sp., USA 
0.99 }— AF 143234, unknown potential host, France H J 
AF 143233, Pinus sp. USA P. tinctorius 
AF 228647, Pinus caribaea, Kenya (P. species 6) 
S5PISOLI, Pinus sp., Spain ; fi 
1.00 AF228645, Quercus ilex/ Q. coccifera, Spain _ 
AF228646, Quercus ilex/Cistus ladanifer, Spain 
AF374625, Pinus kesiya, Thailand 
AF374679, Eucalyptus sp., China 
0.69 9PISOLI, Pinus kesiya, Thailand 
ABopoRt: panknown potential host, Thailand 
inus kesiya, Thailan q . 
TIPISOLI, Pinus kesiya, Thailand P. orientalis 
10PISOLI, Pinus kesiya, Thailand A 
TPISOLI, Pinus kesiva, thailand (P. species 5) 
8PISOLI, Pinus kesiya, Thailand 
AF374711, Pinus sp./Eucalyptus sp., China 
34PISOLI, Pinus kesiya, Thailand 
AB099919, unknown potential host, Thailand 


0.1 


Fic. 2. Phylogenetic tree obtained with a Bayesian Monte Carlo Markov (MCMC) analysis 
assuming HKY + G model, as suggested by MrModeltest 2.3, of ITS nrDNA sequences of Pisolithus 
collections included in TaBLE 1 and sequences obtained from the GenBank and UNITE. Each 
branch is labeled with the DNA isolation code in bold (new sequences) or with the accession 
number including the potential host and the country. Numbers above branches represent posterior 
probabilities values. Clades and species numbers according to previous authors. 


et al. (1999) note that basidiospore morphology should be considered carefully 
as the mature spore ornamentation relies on spore development in which nurse 
cells play a vital role and affect final surface features. Morphological differences 
often have diagnostic importance for new morphological species, (e.g., Astraeus 
odoratus; Phosri et al. 2004, 2007), despite instances of convergence. 


202 ... Phosri & al. 


Sowerby (1814), who provided an excellent illustration of a gasteroid fungus, 
apparently coined the epithet “Lycoperdon capsuliferum based on the fact that 
the fertile part of the fruiting body contained small spore capsules resembling 
a fig fruit. Fries (1829: 54) recognized the significance of this collection and 
how it differed from other species known to him mainly from the classical 
literature. He formally proposed the superfluous new name Polysaccum 
olivaceum in section Radiculosa for Sowerby’s species, differing from the other 
members of the section in spore-mass colour and fruiting body shape. Because 
of the morphological and habitat similarities shared by Sowerby’s fungus and 
modern ‘sp. 14’ clade collection, we are confident in identifying this material as 
Lycoperdon capsuliferum, which we transfer to Pisolithus. 


Pisolithus capsulifer (Sowerby) Watling, Phosri & M.P. Martin, comb. nov. Fic. 3 
MycoBank MB 519949 [PISOLITHUS SP. 14] 


= Lycoperdon capsuliferum Sowerby, Col. Fig. Engl. 
Fung., Suppl.(no. 31): pl. 425a/b. 1814. 


= Polysaccum olivaceum Fr., Syst. Mycol. 3(1): 54. 1829 [nom. nov. superfl.]. 

Type: Coloured plate 425a/b in Sowerby, Col. Fig. Engl. Fung., Suppl.(no. 31), 1814 

(lectotype designated here). ENGLAND, Berkshire, Sandhurst, 7 Sep 1993, leg. E.E. 

Green, Wat. 25310 (epitype designated here, E00290168; GenBank FR748135) 
BASIDIOMES turbinate to pyriform with subglobose to ellipsoid head 40-60 
mm diam. or flattened at the apex > 90 mm broad, tapering into solid, firm, 
irregular pseudostipe incorporating soil debris; pseudostipe 15-30(-90) mm 
long often flattened and then 10-25 mm broad truncated downwards to 15 mm. 
PERIDIUM wafer thin, > 1mm thick, dry, smooth, yellowish ochraceous with 
olivaceous spots and faint netted appearance from outline of pseudoperidioles 
below, then fragmenting to expose powdery spore-mass and disintegrating 
pseudoperidioles. Lower part more compacted and with an olivaceous- 
yellow hue, finally drying hard after weathering to become clinker-like. 
PSEUDOPERIDIOLES pale then red-brown finally burnt umber, larger at apex 
and compressed downwards, becoming pulverulent. 

CLAMP-CONNECTIONS present. BASIDIOSPORES 6.8-8.2-10.6 um, globose, 
spinose spines at first separate and 1.1-1.9-2.9 um long, before collapsing 
to coalesce into groups and falling back onto spore surface to give a rivulose 
effect. 

ADDITIONAL SPECIMENS EXAMINED: FRANCE, 20 km. from Limoges, mixed 
broadleaf-conifer forest, 1-Sep-1997, leg. E.E. Green, Wat. 25298 (E00159822). 
UNITED KINGDOM, Hampsnirg, Ringwood, 12-Sep-1926, coll. E.J.H. Corner 81444 
(E00075037); coll. E.J.H. Corner 81446 (E00075038); BERKSHIRE, Windsor Great Park, 
Jul-1993, leg. EE. Green, Wat. 25311 (E00290167); Caesar’s Camp near Bracknell, Sep- 


1970, leg. E.E. Green (E00159834); 9-Sep- 1973, leg. E.E. Green, Wat. 10118 (E00185017); 
3-Oct-1981, leg. E.E. Green, Wat. 14216 (E00185018). 


Pisolithus orientalis sp. nov. (Thailand) ... 203 


LyS 


Fic. 3. SEM of spore ornamentation of selected Pisolithus collections (bars = 1 pm). a-e: 
P. capsulifer. From England—a-b: epitype E00290168 (Wat. 25310, 46pisoL1), c-d: E00185017 
(Wat. 10118, 49PIsoL1); from France—e: E00159822 (Wat. 25298, 53pIsoLt). f: PB. orientalis. 
Holotype BBH28597 (33PIsoLi). 


ComMENTSs. The distribution of the pseudoperidioles in fresh material is clearly 
expressed by Sowerby (1814) when he wrote, “at first strong and very rugged but 
the congeries of seeds, if I may so-call them, were enveloped or compressed into 
forms laying by each other, giving a reticulated appearance in some directions, 
being mostly rather oblong, and of a dark brown colour...and looks like pollen 
bursting from something analogous to anthers.” 


204 ... Phosri & al. 


Sete 


/ & 426 C Ltda 

‘ Sign Geos Ct E€ehlarcs 

4) b 

Me; éofecicleaeu Cafeticks ye recs Sov 
/ 


¢ 
So laps Hedddrr bet € 
c 


— te 


Fic. 4. a: Sowerby’s illustration of Lycoperdon capsuliferum (arrow). 
b: Pisolithus orientalis Holotype BBH28597 (33PIsoL!). 


Pisolithus orientalis sp. nov. (Thailand) ... 205 


Pisolithus orientalis Watling, Phosri & M.P. Martin sp. nov. Fia. 4 
MycoBank MB519952 | PISOLITHUS SP. 5] 


Differs from Pisolithus tinctorius by smooth pedicellate basidiomata with blackish brown 
discs, smaller globose basidiospores ornamented by isolated groups of narrow cones 
that adhere together to form secondary spines, and an association with Pinus kesiya. 


Type: THAILAND. Chang Mai, Hot District, Bo Kaew pine plantation, under Pinus 

kesiya, 11-Aug-2001 (holotype, BBH 28597; GenBank FR748148). 
BASIDIOME: 35-55 mm high, pedicellate to broadly clavate, divided into 
distinct head and cylindrical stipe, snuff-brown to cigar-brown with a black 
shining surface around the head, surface smooth, not scaly. PERIDIUM: 24-45 
mm broad, broadly ellipsoid to subglobose, rough, thin, brittle, snuff-brown, 
breaking up at the apex to expose powdery contents and join up to stipe. STIPE: 
13-24 x 10-15 mm woody, snuff-brown, smooth, cylindrical base of agglutinate 
mycelium and soil, yellowish rhizomorphs when immature. GLEBA: snuff- 
brown to ferruginous powdery mass formed by the breakdown of peridioles 
seated in a small black pocket within the peridium. PERIDIOLEs: thin-walled, 
ovoid-ellipsoid, smooth, clay-buff, 2 x 1 mm, at first clay-buff then more snuff- 
brown or ferruginous as they mature, discrete, but later breaking down into 
a snuff brown to ferruginous powdery mass, exuding sienna to fulvous fluid 
when mounted in alkaline solution. 

CONSTITUENT HYPHAE: intertwined, hyaline to ochraceous, thin-walled, 
2.4-4.8 mm broad, septate without encrustation, clamp-connections present. 
BASIDIA not seen. BASIDIOSPORES: globose, 5.8-9.0-10.7 um including spines, 
heavy ornamentation consisting of isolated groups of connate, narrow cones 
adhering together to form points, lacking connections. 

ADDITIONALSPECIMENS EXAMINED: THAILAND, Chiangmai, 11-Aug-2001 (BBH28598; 

GenBank FR748149), together with other specimens referred to P orientalis (sp. 5) in 

TABLE 1.Other sequences obtained were directly from cultures. 
The pedicellate, smooth basidiomes that become blackish brown at the disc 
above the first maturing pseudoperidioles, the relatively small spores that 
are covered with narrow cones that join together to form connate secondary 
spines, and an association with Pinus kesiya distinguish Pisolithus orientalis 
from other species in the genus. The distinctive spore ornamentation coupled 
with the molecular data distinguishes this new taxon. The characters emphasis 
how important it is to examine in detail the development of both basidiome 
and basidiospores in Pisolithus. 


Conclusion 

Our study confirms that Pisolithus tinctorius sensu lato, as previously and 
widely understood, covers several separate species. Molecular work linked 
to scanning electron microscope studies of the basidiospores is proving 


206 ... Phosri & al. 


essential in unravelling speciation within Pisolithus. The marriage of data from 
classical sources and our molecular and morphological studies confirms the 
distinctiveness of the British, French, and Swedish collections and a new species 
from SE Asia growing with Pinus kesiya. 


Acknowledgements 

We thank herbaria BCN, E for loan of specimens used and A.D. Parker for material 
from his personal herbarium. Part of the work was supported by the Ministerio de 
Educacién y Ciencica (CGL2006-12732-CO2-01/BOS). CP is indebted to the National 
Research Council of Thailand (NRCT) and Royal Thai Government for financial support 
and to the European Commission Human Potential Programme for supporting part of 
this study at the Real Jardin Botanico de Madrid (BIODIBERIA). Authors also thank 
peer reviewers for comments and suggestions. 


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ISSN (print) 0093-4666 © 2012. Mycotaxon, Ltd. ISSN (online) 2154-8889 


MY COTAXON 


http://dx.doi.org/10.5248/120.209 
Volume 120, pp. 209-213 April-June 2012 


Terriera simplex, a new species of Rhytismatales from China 


X1A0-MING Gao! CHUN-TAO ZHENG! & YING-REN LIN” 


' School of Life Science & ? School of Forestry & Landscape Architecture, 
Anhui Agricultural University, West Changjiang Road 130, Hefei, Anhui 230036, China 
*CORRESPONDENCE TO: *yingrenlin@yahoo.com 


ABSTRACT —A new Terriera species found on fallen leaves of Trachelospermum jasminoides 
(Apocynaceae) is described, illustrated, and designated as Terriera simplex. This taxon is 
distinguished from its closest relatives by unbranched paraphyses and asci with truncate 
or subtruncate apices. The type specimen is deposited in the Reference Collection of Forest 
Fungi of Anhui Agricultural University, China (AAUF). 


KEY worpDs —taxonomy, Ascomycota, morphology, vine plant 


Introduction 

The genus Terriera B. Erikss. was established by Eriksson (1970) for T. clado- 
phila (Lév.) B. Erikss., belonging to Rhytismataceae (Kirk et al. 2008). Minter 
(1996) provided a detailed description of the type species. 

Over the years, however, some species which should belong to Terriera have 
been mistakenly placed in Clithris (Fr.) Bonord., Dermascia Tehon, Hypoderma 
De Not., and (particularly) Lophodermium Chevall. (Johnston 2001). Johnston 
(2001), who divided Lophodermium on monocotyledonous plants into groups 
A, B, C, and D, regarded Group B species as closer to the type species of Terriera 
based on their special ascomatal structure. He therefore transferred 11 species 
and 1 variety of Lophodermium Group B to Terriera as new combinations 
and published three new species. The author also invalidly published a fourth 
species, in that work, which he later validly published as T! samuelsii P.R. 
Johnst. (Johnston 2003). In the paper by Ortiz-Garcia et al. (2003), Johnston 
recombined Clithris minor Tehon as T: minor (Tehon) PR. Johnst. rDNA-ITS 
sequence analyses by Ortiz-Garcia et al. (2003) indicated that Lophodermium 
from pine hosts were distally related to Terriera and that two monophyletic 
Terriera sequences formed a weakly supported sister group related to Lirula 
macrospora (R. Hartig) Darker. 


210 ... Gao, Zheng & Lin 


Five Terriera species have previously been reported from China: T! brevis 
(Berk.) P.R. Johnst., T. huangshanensis Z.Z. Yang et al., T. camelliae (Teng) 
Y.R. Lin & Jiang L. Chen, T’ coacervata Y.R. Lin & Q. Zheng, and T: illiciicola 
(S.J. Wang et al.) Q. Zheng & Y.R. Lin (Frohlich & Hyde 2000, Yang et al. 2011, 
Chen et al. 2012, Zheng et al. 2012). In the present paper, we describe a new 
species of Terriera on confederate jasmine, a vine from the Dabie Mountains in 
Anhui Province, China. 


Materials & methods 

External characteristics of conidiomata and ascomata obtained from open ascomata 
of the holotype specimen were observed under a dissecting microscope at 10-50x. 
After rehydration in water for 10 min, 10-15 um thick sections of the conidiomata 
and ascomata were cut using a freezing microtome. Microscopic examinations were 
made in water, 5% KOH, Melzer’s reagent, or 0.1% (w/v) cotton blue in lactic acid. For 
observing the outlines of ascomata and conidiomata in vertical section, sections were 
mounted in lactic acid or cotton blue with pretreatment in water. Gelatinous sheaths 
surrounding ascospores and paraphyses were observed in water or cotton blue in lactic 
acid. The color of internal structures and ascospore contents were observed in water. 
Measurements and drawings of asci, ascospores, and paraphyses (30 per specimen) were 
made using material mounted in 5% KOH or Melzer’s reagent. Point and line integrated 
illustrations of external shapes and internal structures of the conidiomata and ascomata 
were prepared using a microscope drawing device. 


Taxonomy 


Terriera simplex Y.R. Lin, X.M. Gao & C.T. Zheng, sp. nov. FIGs 1-6 
MycoBank MB 563610 


Differs from T: cladophila in unbranched paraphyses and asci with truncate or 
subtruncate apices and from T. minor by the presence of conidiomata and ascomata 
with obtuse, rounded or slightly acute ends. 


Type: on fallen leaves of Trachelospermum jasminoides (Lindl.) Lem. (Apocynaceae), 
China, Anhui, Dabie Mountains, Tiantangzhai, alt. ca 700 m, 16 September 2005, 
S.J. Wang and Y.R. Lin 2020 (Holotype AAUF 68128). 


EryMo_oey: simplex, referring to the simple paraphyses, which are not branched near 
the apex. 
ZONE LINES absent. 
Conipi1omarta on both sides of leaves, crowded or sometimes coalescent. 
In surface view, conidiomata 130-200 um diam., rounded or subrounded, 
black-brown in the centre, more or less concolorous with the substratum surface 
elsewhere, slightly raising the leaf surface, discharging spores through a 3-5 um 
diam. apical ostiole. In vertical section, conidiomata subepidermal, lenticular. 
UPPER WALL very poorly developed, composed of tiny, thin-walled globose cells 
1.6—2 um diam. BASAL WALL well developed, 12-17 um thick, consisting of 2-4 
(-5) layers of angular cells 2—4.5 um diam. SUBCONIDIOGENOUS LAYER 4.5-8 


Terriera simplex sp. nov. (China) ... 211 


pa Ky . 
os wie dgreleos 

OG Fer a TOSS 
SA 40 Sy OS 
pice ke eaactd 


Fics 1-6. Terriera simplex on Trachelospermum jasminoides. 1. A leaf bearing fruit bodies. 
2. Conidiomata and ascomata observed under a dissecting microscope. 3. Ascoma in median 
vertical section. 4. Portion of ascoma in median vertical section. 5. Paraphyses, asci and ascospores. 
6. Conidioma in vertical section. 


2D ae: Zheng & Lin 


um thick, composed of textura angularis with nearly colorless, thin-walled 
cells. CONIDIOGENOUS CELLS 6-11 x 2-3 um, subcylindrical, tapering to the 
apex, proliferating sympodially. Conrp1a 2.5—4 x ca 1 um, cylindrical or nearly 
elliptical, hyaline, unicellular. 

Ascomarta in similar positions to conidiomata on the substratum, crowded 
in irregular, light yellow to grayish-white, large bleached areas without 
obvious edges, sometimes 2—6 ascomata coalescent. In surface view, ascomata 
650-1000 x 350-480 um, elliptical to ovate, ends obtuse, rounded or slightly 
acute, black, slightly shiny, with a clearly marked outline, moderately raising 
the surface of the leaf, opening by a single longitudinal split which is sometimes 
branched in the triangular ascomata. Immature ascomata seen as two parallel 
black patches on the substratum surface rather than as a single more or less 
elliptical one, with a pale central zone along future line of opening. Lips absent; 
split extending 3/4—-4/5 the length of the ascoma. In median vertical section, 
ascomata subepidermal, with epidermal cells becoming filled with fungal tissue 
as ascomata develop, 120-135 um deep. COVERING STROMA 18-22 um thick 
near the opening, black-brown, composed of textura angularis with thick- 
walled cells 3-5.5 um diam. Along the edge of the ascoma opening, there is 
a flattened, 8-12 um thick extension adjacent to the covering stroma, and 
which comprises strongly carbonized tissue with no obvious cellular structure. 
EXCIPULUM very poorly developed, arising from the inner layer of the basal 
stroma, consisting of hyaline textura porrecta. BASAL STROMA 6-15 um thick, 
dark-brown, composed of 2—3(—4) layers of 3-5 um diam., angular, thick- 
walled cells. 20-32 um thick textura prismatica with somewhat thin-walled, 
nearly colorless to grayish-brown cells exists between the covering stroma and 
basal stroma. SUBHYMENIUM moderately developed, 7-12 um thick, consisting 
of hyaline textura angularis and intricata. PARAPHYSES 95-110 x 1.2-1.5 um, 
filiform, aseptate, not branched, occasionally gradually or abruptly swollen to 
2.5-3 um near the apex where sometimes agglutinated, covered in a ca 1 um 
thick gelatinous matrix. Asci ripening synchronously, 72-95(-105) x 4.8-5.2 
uum, cylindrical, very short-stalked, thin-walled, apex truncate, subtruncate or 
obtuse, without circumapical thickening, J-, 8-spored. AscospoREs arranged 
in a fascicle, (45—)56-82 x 1-1.2 um, filiform, slightly tapered towards the 
rounded base, hyaline, with a gelatinous sheath 0.8-1 um thick. 

HOsT SPECIES & DISTRIBUTION: producing conidiomata and ascomata 
on fallen leaves of Trachelospermum jasminoides; known only from the type 
locality, Anhui, China. 


COMMENTS—We place our new species in Terriera based on the presence of 
a strongly carbonized extension adjacent to the ascoma opening and textura 
prismatica in the corner between the covering and basal stroma and the absence 
of lip cells. 


Terriera simplex sp. nov. (China) ... 213 


Terriera simplex is distinguished from the type species T: cladophila by its 
sometimes coalescent and occasionally three-lobed ascomata, paraphyses with 
unbranched upper parts, and asci with truncate or subtruncate apices. While 
ascomata in T: cladophila are scattered and sometimes circular, the paraphyses 
are frequently and irregularly branched and irregularly twisted or swollen at 
the apex, and the tips of asci are rather rounded (Minter 1996). Terriera minor, 
a widely distributed species, resembles T: simplex but differs in ascomata with 
rounded ends that are not associated with conidiomata, paraphyses that branch 
2-3 times in the upper 30-40 um, and sequentially maturing asci (Johnston 
1988, 1989a,b). 

Ascomata of T: simplex are mature on dead leaves. It is not known whether 
this fungus occurs in living leaves as a mutualistic endobiont or as a parasite. 
This needs to be investigated. 


Acknowledgments 

We thank the National Natural Science Foundation of China (No. 30870014) and 
the Specialized Research Fund for the Doctoral Program of Higher Education of China 
(No. 20070364002) for financial support. Thanks are also given to Dr D.W. Minter and 
Dr M. Ye for pre-submission reviews leading to the improvement of our manuscript, 
and to Dr S.J. Wang for the field investigations. 


Literature cited 

Chen JL, Lin YR, Hou CL, Wang SJ. 2012 [“2011”]. Species of Rhytismataceae on Camellia spp. 
from the Chinese mainland. Mycotaxon 118: 219-230. http://dx.doi.org/10.5248/118.219 

Eriksson B. 1970. On Ascomycetes on Diapensales and Ericales in Fennoscandia. Symb. Bot. Upsal. 
19: 1-71. 

Frohlich J, Hyde KD. 2000. Palm microfungi. Fungal Diversity Press. Hong Kong. 364 p. 

Johnston PR. 1988. An undescribed pattern of ascocarp development in some non-coniferous 
Lophodermium species. Mycotaxon 31: 383-394. 

Johnston PR. 1989a. Lophodermium (Rhytismataceae) on Clusia. Sydowia 41: 170-179. 

Johnston PR. 1989b. Rhytismataceae in New Zealand 2. The genus Lophodermium on indigenous 
plants. New Zealand J. Bot. 27: 243-274. 

Johnston PR. 2001. Monograph of the monocotyledon-inhabiting species of Lophodermium. 
Mycol. Pap. 176: 1-239. 

Johnston PR. 2003. Validation of Terriera samuelsii. Mycotaxon 87: 1-2. 

Kirk PM, Cannon PF, Minter DW, Stalpers JA. 2008. Ainsworth & Bisby’s dictionary of the fungi, 
10" ed. CAB International. Wallingford. 771 p. 

Minter DW. 1996. Terriera cladophila. IMI Descr. Fungi & Bact. no. 1296. 

Ortiz-Garcia $, Gernandt DS, Stone JK, Johnston PR, Chapela IH, Salas-Lizana R, Alvarez-Buylla 
ER. 2003. Phylogenetics of Lophodermium from pines. Mycologia 95: 846-859. http://dx.doi. 
org/10.2307/3762013 

Yang ZZ, Lin YR, Hou CL. 2011. A new species of Terriera (Rhytismatales, Ascomycota) from 
China. Mycotaxon 117: 367-371. http://dx.doi.org/10.5248/117.367 

Zheng Q, Lin YR, Yu SM, Chen L. 2012 [“2011”]. Species of Rhytismataceae on Lithocarpus spp. 
from Mt Huangshan, China. Mycotaxon 118: 311-323. http://dx.doi.org/10.5248/118.311 


ISSN (print) 0093-4666 © 2012. Mycotaxon, Ltd. ISSN (online) 2154-8889 


MY COTAXON 


http://dx.doi.org/10.5248/120.215 
Volume 120, pp. 215-221 April-June 2012 


The Leptogium juressianum complex in southeastern Brazil 


Marcos J. KITAURA* & MARCELO P. MARCELLI? 


‘UNESP, Instituto de Biociéncias, Depto de Botanica, 

Distrito de Rubido Jr., Caixa Postal 510, Botucatu/SP, CEP 18618-970, Brazil 
?Instituto de Botanica, Nucleo de Pesquisa em Micologia, 

Caixa Postal 68041, Sdo Paulo/SP, CEP 04045-972, Brazil 


*CORRESPONDENCE TO: junjimjk@gmail.com 


AsBstTRACcT— Leptogium subjuressianum and L. subjuressianum var. caparoense are new 
Brazilian taxa growing mostly in the southeastern mountains. They are differentiated from 
European taxa by the ornamentation of the upper surface. 


Key worps— lobules, margin, granular isidia 


Introduction 

Leptogium juressianum is the only species in Leptogium sect. Mallotium 
producing isidia-like structures and with hairs confined mostly to the upper 
surface (Jorgensen 1997, Tavares 1950). The other Leptogium species with 
hairs chiefly on the upper surface — L. eriodermoides Arv. & P.M. Jorg. and 
L. resupinans Nyl. Jorgensen 1997) — do not have ornamented thalli. 

Such hairs, initially observed by Tavares (1950), are commonly considered 
an important taxonomic characteristic and have a specific organization. 
However, the organization is rarely described in detail and sometimes not even 
mentioned (Swinscow & Krog 1988, Galloway & Jorgensen 1995). 

Jorgensen & James (1983) described the hairs of L. juressianum as an 
arachnoid tomentum while Jorgensen (1997) called them cobwebby hairs. 
Tavares (1950) described the holotype from Portugal as producing isidiate or 
lacinulate propagules, but later specimens with alternative propagules were 
also identified as L. juressianum: cylindrical to complanate isidia and lacinulae 
were noted for Ethiopian (Africa) collections (Swinscow & Krog 1988), western 
European specimens produced granular to sublobulate isidia Jorgensen & 
James 1983), and the upper surface of specimens from Chile were found to have 
granular, sublobulate, or lacinulate isidia and isidiate-lacerate lobe margins 
(Galloway & Jorgensen 1995). 


216 ... Kitaura & Marcelli 


Leptogium specimens collected during the first author's doctoral studies, 
were initially identified as L. juressianum. Upon type revision and under our 
descriptive protocol we discovered that the material represents new taxa, which 
are described here, together with comments on L. juressianum. 


Material & methods 

This study is mainly based on material collected in the Serra da Mantiqueira, a major 
mountain complex in southeastern Brazil, as well as on the isotype of L. juressianum 
(UPS). We were unable to study the holotype because the Museu Nacional de Historia 
Natural (LISU) in Lisboa, Portugal, did not respond to a loan request. 

Comparative morphological and anatomical characteristics summarized in TABLE 1 
are modeled after a table first assembled by Cunha (2007), who studied the Leptogium 
species collected in forests (mainly mangroves) and urban littoral habitats of Sao Paulo 
state. The character set has been modified to encompass the morphological variations 
we observed in the Serra da Mantiqueira taxa. 

The nature of the thalline surfaces and their ornamentation are considered important 
taxonomic characters. The surfaces are described without and with magnification; the 
system of folds and/or wrinkles and their size is determined by complex organization 
of the hyphae (both cortical and internal), which remain constant in both dry and wet 
specimens. Some folds are readily seen with the naked eye, but smaller folds (< 0.1 mm, 
which appear smooth to the naked eye but can readily be seen under magnification = 
10x) must be observed under the stereomicroscope. 

Several species apparently bear similar propagules on the upper surface, but careful 
observation has shown that their morphology and position are good species characters. 
Thus descriptions of their form, dimensions, distribution, and ontogeny are described 
in TABLE 1. 


TABLE 1. Morphological comparison of Leptogium juressianum, L. subjuressianum, 
and L. subjuressianum var. caparoense. 


TAXON ORNAMENTATION ORNAMENT HAIR Dry THALLUS 
DISTRIBUTION DISTRIBUTION COLOR* 

L. juressianum Cylindrical Usually on Upper surface; Gray to bluish 
when young to lobe margins parts of where glabrous 
flattened when underside 
fully developed 


L. subjuressianum 


var. subjuressianum 


L. subjuressianum 
var. caparoense 


Granular isidia 
and lobules 


Granular isidia 


* All whitish where hairy 


Usually on 
lobe margins 


Abundant on 
lamina; frequent 
on lobe margins 


Upper surface 
and usually 
on underside 
margin 


Frequent on 
upper surface; 
abundant on 
underside 


Brownish where 
glabrous 


Dark brown to 
blackish where 
glabrous 


Leptogium subjuressianum sp. nov. (Brazil) ... 217 


All specimens analyzed were dissected under a stereomicroscope with a razor blade 
and the semi-permanent slides were mounted with solution of 50% glycerine. 


Results & discussion 

The name Leptogium juressianum has been attributed to all ornamented 
(bearing isidia-like or lobule-like structures) specimens of Leptogium that 
develop hairs mostly on the upper surface (Tavares 1950, Jorgensen & 
James 1983, Jorgensen & Nash 2004). The detailed descriptions of thallus 
and ornamentation produced according to our protocols have enabled us to 
distinguish the Brazilian taxa. This was not possible using brief descriptions and 
the characters commonly used to describe and differentiate Leptogium species. 
Thallus color, hair distribution, and the nature of the vegetative propagules are 
the principal diagnostic characters in this paper (TABLE 1). 


Leptogium juressianum Tav., Port. Acta Biol., Ser. B, 3: 68, 1950. FIGs 1-2 
Type: PORTUGAL, Minho [corresponding with the modern districts of Braga and 
Viana do Castelo], Serra do Géres, between Pedra Bela and the Caldas, 700 m alt., on 
Arbutus unedo, 16.V1.1947, leg. C. Tavares 2081 (holotype, LISU; isotype, UPS!). 

THALLUS gray, Opaque, matt, gray to whitish when observed under the 

stereomicroscope. Loses irregularly lacerate, to 3 mm wide, overlapping, 

attached in points, convolute, upper surface smooth and velvety under the 
naked eye, pubescent at 10x and higher magnifications (interlaced hairs that 
are similar to sponge fibers); apices rounded, usually ascending to involute, 
smooth or isidiate to lacinulate; lateral margin fimbriate (isidiate to lacinulate), 
ascending, irregular; lower side bluish gray, smooth at different magnifications. 
Istp1A absent. LACINULES smooth, cylindrical when young to flattened, ca. 
0.08-0.30 x 0.03-0.08 mm, unbranched to irregularly branched but not coralloid, 
erect, firm, concolorous with the thallus, usually marginal, dense, frequent to 
abundant, grouped on the lamina. ATTACHED by hairs; rhizines absent; hapters 
absent; hairs irregular, unbranched or branched, up to 125 um (ca. 9 cells) long, 
dense on upper surface, frequent on lower side. APOTHECIA absent. 
ANATOMY— THALLUS ca. 60 um thick, quadratic cells of the cortices ca. 

7.5 x 5.0 um; columnar hyphae ca. 2.5 um thick, straight to inclined, 2(-3) 

cells. CYANOBACTERIA blue, frequent to abundant, cell number not appraised, 

spherical, 5 um diam.; gelatinous matrix poor, hyaline. Pycnip1a absent. 


CoMMENTS— Leptogium juressianum is characterized by the isidiate to 
lacinulate margins and hairs on the upper surface. 

Originally described from Portugal, the species has been cited for western 
Europe (Jorgensen & James 1983), southern Africa (Swinscow & Krog 1988), 
and Chile, South America (Galloway & Jorgensen 1995), but always with 
different type and/or position of propagules. Since the propagules are major 


218 ... Kitaura & Marcelli 


diagnostic characters, we believe the previously cited collections may represent 
different taxa and that all that material needs re-evaluation 


Leptogium subjuressianum Marcelli & Kitaura, sp. nov. Fics 3-4 

MycoBAank 563829 

Differs from Leptogium juressianum by granular isidia and presence of rounded lobules. 

TyPE: Brazil, Rio Grande do Sul State, municipality of Tapes, on trunk of a tree, 5 m alt, 

29.1.1994, leg. M.P. Marcelli 26459 (holotype, SP). 

ErymMo oey: The specific epithet refers to the closely related species, L. juressianum, the 

name first applied to the studied material. 
THALLuS whitish or black (naked eye), opaque, matt, whitish brown when 
observed under the stereomicroscope. LoBEs to 2 mm wide, irregularly 
overlapping, attached in points, ascending; upper surface velvety when observed 
with the naked eye, pubescent at 10x and higher magnifications; apices rounded, 
ascending, involute to convolute, ornamented with small granular to lobulate 
isidia; lateral margins lobulate, ascending or flat, undulate; lower side bluish 
gray, smooth, covered by long hairs seen at 10x and higher magnification. 
Isip1a granular, ca. 0.05 mm diam., unbranched, erect, firm, concolorous with 
the thallus, rare on the lamina but dense at the margins. LoBuLEs with apices 
rounded, ca. 0.5 mm diam., unbranched, erect, firm, concolorous with the 
thallus, marginal, dense, abundant. ATTACHED by rhizines and hairs; hapters 
absent; rhizines unbranched, beige, evenly distributed, frequent, formed from 
agglutinated hairs; hairs interlaced, unbranched or branched, < 125 um (ca. 8 
cylindrical cells) long, beige, present on both surfaces, abundant on the upper 
surface and frequent on the lower surface. APOTHECIA absent. 

ANATOMY— THALLUS ca. 55 um thick, upper quadratic cells ca. 10 x 10 
um, lower quadratic cells ca. 5 x 5 um, columnar hyphae ca. 2.5 um thick, 
straight and inclined, comprising 3-4 cells. CYANOBACTERIA blue, frequent, 
cell number not appraised, cells spherical, ca. 5 um diam.; gelatin frequent, 
colorless. PycNIDIA absent. 

ADDITIONAL MATERIAL EXAMINED: BRAZIL. PARANA STATE, General Carneiro 
Municipality, Fazenda Lageado Grande, s/d, S. Eliasaro 2892 (UPCB); SAo PAULO 
STATE, Campos do Jordao Municipality, Parque Estadual de Campos do Jordao, 
Araucaria/Podocarpus forest, on tree trunk inside the forest, 1400 m alt., 17.V1.1995, M.P. 
Marcelli, A. Gugliotta & R. Maziero 28914 (SP); MINAS GERAIS STATE, Camanducaia 
Municipality, on trunk, 21.X1.2008, M.J. Kitaura, M.P. Marcelli, A.E. Luchi & S.N. Inoue 
1158 (SP), 1179 (SP); 22.X1.2008, M.J. Kitaura, M.P. Marcelli, A.E. Luchi & S.N. Inoue 
1232 (SP), 1235 (H); Itamonte Municipality, BR-485, on trunk, 22.1.2009, M.J. Kitaura 
& M.P. Marcelli 1468 (SP); Brejo da Lapa, on trunk, 23.1.2009, M.J. Kitaura & M.P. 
Marcelli 1536 (SP); Espigtro SANTO STATE, Dores do Rio Preto Municipality, on trunk, 


16.1X.2009, M.J. Kitaura & M.P. Marcelli 1792 (SP), 1804 (SP), 1825 (BOTU); on branch 
fallen on the rock, 16.1X.2009, M.J. Kitaura & M.P. Marcelli 1911 (SP). 


Leptogium subjuressianum sp. nov. (Brazil) ... 219 


FiGuRES 1-6. Leptogium juressianum (isotype, UPS): 1. Thallus; 2. Propagules. L. subjuressianum 
var. subjuressianum (holotype): 3. Thallus; 4. Propagules. L. subjuressianum var. caparoense 
(holotype): 5. Thallus; 6. Propagules. Scale bars: 1, 3, 5 = 5 mm; 2, 4, 6 = 20 um. 


220 ... Kitaura & Marcelli 


ComMMENTS— Leptogium subjuressianum is characterized by the rounded 
lobules on the thallus margin and spongioid hairs on the upper surface, which 
usually appears whitish due to the presence of dense hairs. Both L. juressianum 
and L. subjuressianum are covered by a dense layer of spongioid hairs, which 
are interlaced and resemble fibers of sponges. 

According to Tavares (1950), L. juressianum possesses only isidia and 
lacinules on the margins. No rounded lobules were observed in any part of 
the isotype. We did not find lacinules in any specimens of L. subjuressianum, 
where only granular to lobulate isidia are present prior to the development of 
lobules. 

Leptogium subjuressianum is common in Brazil and is usually collected on 
tree trunks. Specimens were collected from Tapes Municipality, Rio Grande 
do Sul State (30°40'12"S 51°23'24"W) to Alto Caparaé Municipality in Minas 
Gerais State (20°29'42"S 41°59'54"W). 


Leptogium subjuressianum var. caparoense Kitaura & Marcelli, var. nov. 
MycoBAnkK 563862 FIGs 5-6 


Differs from Leptogium subjuressianum var. subjuressianum by the isidia distribution 
and the presence of rhizomorphic hyphae on the lower surface. 


Type: Brazil, Minas Gerais State, Municipality of Alto Caparad, Cachoeira Bonita, 
on organic material on rock beside the waterfall, 17.1X.2009, leg. M.J. Kitaura & M.P. 
Marcelli 1900 (holotype, SP; isotypes, H). 


Erymo.oey: ‘The varietal epithet refers to the Caparad Range, a major mountain 
complex in southeastern Brazil where the types were collected. 

THALLUS whitish to blackish (naked eye), opaque, matt, whitish (hairy part) and 
dark brown or black (glabrous part) when observed under the stereomicroscope. 
Loses to 3 mm wide, irregular, overlapping, attached in points, ascending, 
upper surface smooth or velvety when observed with the naked eye, pubescent, 
smooth or ornamented at 10x and higher magnifications; apices rounded, 
ascending or involute, usually smooth; lateral margins smooth to granular, 
ascending, undulate; lower side usually brownish, with adhering substrata when 
observed with the naked eye, smooth or less pubescent that upper surface at 10x 
and higher magnifications. Isrp1a granular, ca. 0.1 mm diam., concolorous with 
the thallus, firm, dense or abundant on the lamina and frequent at the margins. 
LOBULES rounded, ca. 0.5 mm diam., unbranched, erect, firm, concolorous 
with the thallus, marginal when the thallus is covered by hairs, frequent or 
rare on the lamina. ATTACHED by rhizines and hairs; hapters absent; rhizines 
unbranched, beige, evenly distributed, frequent, constituted from agglutinated 
hairs; hairs interlaced, irregular, beige, dense on upper surface and frequent on 
lower surface, cells cylindrical. APOTHECIA absent. 

ANATOMY— THALLUS < 70 um thick, upper quadratic cells (wall slightly 
blackish) ca. 10 x 10 um, lower quadratic cells ca. 7.5 x 7.5 um; columnar hyphae 


Leptogium subjuressianum sp. nov. (Brazil) ... 221 


ca. 2.5 um thick, straight to inclined, 4-celled. CYANOBACTERIA yellow to slight 
blue, frequent, cells spherical, ca. 5 um diam.; gelatin frequent to scarce, yellow 
next to upper cortex and colorless below. Pycnip1A absent. 
ADDITIONAL MATERIAL EXAMINED: BRAZIL, Minas GERAIS STATE, Alto Caparad 
Municipality, Cachoeira Bonita, saxicolous, 17.1X.2009, M.J. Kitaura & M.P. Marcelli 


1902 (H), 1905 (SP); on the moss on the rock, 17.[X.2009, M.J. Kitaura & M.P. Marcelli 
1904 (SP), 1932 (BOTU). 


ComMMENTS— Leptogium subjuressianum var. caparoense is characterized by 
granular isidia on both the lamina and margins (older parts) and the blackish 
or dark brown colored thallus (when not covered by spongioid hairs). 

The lobules common on the margins of L. subjuressianum var. subjuressianum 
were not observed in L. subjuressianum var. caparoense (TABLE 1). 

Furthermore, the isidia in L. subjuressianum var. caparoense are mainly 
laminal but primarily marginal in L. subjuressianum var. subjuressianum. Also, 
L. subjuressianum var. caparoense has rhizomorphic hyphae covering the entire 
lower surface whereas in var. subjuressianum they are present mainly at the 
margins of the lobes. 

All specimens of L. subjuressianum var. caparoense were collected in an 
especially humid place on wet rocks beside a waterfall inside Caparaé National 
Park. We attribute to this the abundance of rhizomorphic hyphae and spreading 
of the propagules and hairs and prefer to retain the taxon at the varietal level. 


Acknowledgments 

The authors wish to thank Dr. J.A. Elix and Dr. R. Liicking for the critical revision of 
the manuscript and valuable suggestions. M. Kitaura is grateful to FAPESP (2008/51072- 
3) for a PhD grant and M.P. Marcelli to CNPq for a research grant. 


Literature cited 

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de Sao Paulo. UNESP, Instituto de Biociéncias (Master Thesis), Botucatu. 101 p. 

Galloway DJ, Jorgensen PM. 1995. The lichen genus Leptogium (Collemataceae) in southern 
Chile, South America. 227-247, in: FJA Daniels et al. (eds). Flechten Follmann. Contribution 
to Lichenology in Honour of Gerhard Follmann. Geobotanical and Phytotaxonomical Study 
Group, Botanical Institute, University of Cologne, Germany. 

Jorgensen PM. 1997. Further notes on hairy Leptogium species. Symbolae Botanicae Upsalienses 
32(1): 113-130. 

Jorgensen PM, James PW. 1983. Studies on some Leptogium species of western Europe. Lichenologist 
132109125, 

Jorgensen PM, Nash III TH, 2004. Leptogium. 330-350, in: TH Nash III et al. (eds). Lichen Flora 
of the Great Sonoran Desert Region. Vol. 2, Lichens Unlimited, Arizona State University, 
Arizona. 

Swinscow TDV, Krog H. 1988. Macrolichens of East Africa. British Museum (Natural History). 
London. 

Tavares CN. 1950. Liquenes da Serra do Géres. Portugaliae Acta Biologica, Ser. B, 3: 1-189. 


ISSN (print) 0093-4666 © 2012. Mycotaxon, Ltd. ISSN (online) 2154-8889 


MY COTAXON 


http://dx.doi.org/10.5248/120.223 
Volume 120, pp. 223-230 April-June 2012 


Coriolopsis psila comb. nov. (Agaricomycetes) and 
two new Coriolopsis records for Brazil 


GEORGEA S. NOGUEIRA-MELO’, PRISCILA S. DE MEDEIROS’; 
ALLYNE C. GoMES-SILVA’, LEIF RYVARDEN?, HELEN M.P. SOTAO? 
& TATIANA B. GIBERTONI’ 


* Universidade Federal de Pernambuco, Departamento de Micologia 
Av. Nelson Chaves s/n, CEP 50760-420, Recife, PE, Brazil 
*Museu Paraense Emilio Goeldi, Coordenagao de Botanica, 
Caixa Postal 399, CEP 66040170, Belém, PA, Brazil 
>University of Oslo, Department of Botany, P. O. Box 1045, Blindern, N-0316, Oslo, Norway 


*CORRESPONDENCE TO: georgeacomea@hotmail.com 


AsBstTRACT — The new combination Coriolopsis psila is proposed and C. brunneoleuca and 
C. hostmannii are reported as new to Brazil. Descriptions of these collections and a key to the 
eight accepted Coriolopsis species reported from Brazil are provided. 


Key worps — Polyporaceae, diversity, C. byrsina, C. caperata, C. floccosa 


Introduction 

Coriolopsis Murrill (Murrill 1905) comprises about 17 valid species (Kirk et al. 
2008). The genus is cosmopolitan, although several species are restricted to the 
temperate or tropical zones (Ryvarden & Johansen 1980, Ryvarden & Gilbertson 
1993, Nunez & Ryvarden 2001, Dai 2011, Dai et al. 2011). It is characterized by 
the annual (rarely perennial) pileate or sessile (rarely resupinate) basidiomata 
with great color variation. The hyphal system is trimitic with clamp connections 
on generative hyphae, cystidia are absent, and basidiospores are cylindrical, 
smooth, thin-walled, non-amyloid, oblong—ellipsoid, and hyaline to pale brown 
(Ryvarden & Johansen 1980, Ryvarden 1991, Ryvarden & Gilbertson 1993). 

Only seven Coriolopsis species have previously been reported from Brazil: 
C. aspera (Jungh.) Teng, C. badia (Berk.) Murrill, C. burchellii (Berk. ex Cooke) 
Ryvarden, C. byrsina, C. caperata, C. floccosa, and C. gallica (Fr.) Ryvarden 
(Ryvarden 1988, Gugliotta & Abrahao 2011). 

To increase the knowledge of polypore diversity in Brazil, we record two 
additional species, propose a new combination, and provide identification keys 
to all known Brazilian species. 


224 ... Nogueira-Melo & al. 


Material & methods 

In the Brazilian Amazonia, field trips were undertaken in Para State from July 2006 
to February 2008 in the Floresta Nacional de Caxiuana (1°42'3"S 51°31'45"W) and in 
Rondonia State from 2007 to 2008 in the Estagao Ecoldgica de Cunia (8°04'S 63°31’ W) 
and Parque Natural Municipal de Porto Velho (8°45'S 63°54’W). In Pernambuco 
State, Brazilian mangroves were surveyed from March 2009 to March 2010 in Maria 
Farinha (7°51'24.8"S 34°50'32.7"W), Itamaraca (7°46'52.6"S 34°52'53.3"W), Maracaipe 
(8°32'22.8"S 35°00'29.1” W), and Rio Formoso (8°41'20.8"S 35°06'06.6" W). 

Both recent collections and specimens deposited in INPA, O, RB, and SP were 
studied. Specimens were analyzed macro- (shape, color, hymenial surface) and 
micromorphologically (hyphal system, presence/absence and measurements of sterile 
structures and basidiospores). Slides were prepared with 5% KOH (stained with 1% 
aqueous phloxine) or Melzer’s reagent (Ryvarden 1991). Color designation followed 
Watling (1969). The material was incorporated to HFSL, MG, O, and URM. 


Results & discussion 

After identification of new collections and revision of herbaria, eight 
Coriolopsis species are reported for Brazil. The presence of C. gallica is not 
confirmed, as previous reports actually represent Hexagonia hydnoides (Sw.) 
M. Fidalgo (Tavares 1939, URM 608/IPA 281) or Trametes sp. (Bononi et al. 
1984, SP 156751). Coriolopsis badia is a dubious species because it is considered 
a synonym of either Phellinus badius (Cooke) G. Cunn. (http://www.cbs.knaw. 
nl, http://www.mycobank.org) or C. aspera (Ryvarden & Johansen, 1980). The 
only record of C. badia in Brazil (Gibertoni et al. 2004, URM 77849) represents 
a Trametes sp. 

Coriolopsis brunneoleuca and C. hostmannii are new occurrences for the 
country. Coriolopsis psila, here transferred from Fomes, is probably a new 
occurrence for the Brazilian Amazonia. Coriolopsis byrsina represents a new 
record for the states of Amazonas, Rondénia, and Roraima, C. caperata for 
Amazonas, and C. floccosa for Acre, Amazonas, and Mato Grosso. These new 
occurrences were studied also from collections deposited in INPA, RB and 
SP, underscoring the importance of herbarium revisions and accessibility of 
herbaria records to the knowledge of fungal diversity. 


Coriolopsis brunneoleuca (Berk.) Ryvarden, Norw. Jl Bot. 19: 230 (1972). 
BASIDIOMATA annual, effused-reflexed, gregarious, coriaceous, < 5.5 cm, 
1.5 cm wide and to 1 mm thick. ABHYMENIAL SURFACE tomentose, dull, cigar 
brown (16) to fuscous black (36) with concentric zones of dark color. MARGIN 
entire, acute, concolorous with the abhymenial surface. Context homogeneous, 
fibrous, < 100 um thick, milky coffee (28) to cinnamon (10). HYMENIAL SURFACE 
with pores angular, 2-3 per mm, dissepiments 50-100 um thick, snuff brown 
(17) to clay buff (32). HyPHAL sysTEM trimitic; generative hyphae hyaline, 
branched, clamped, thin-walled, 1.5-2.5 um diam.; skeletal hyphae yellowish to 


Coriolopsis in Brazil... 225 


pale brown, thick-walled, 4-7.5 um diam., dextrinoid; binding hyphae golden- 
yellow, almost solid, 3-5 um diam. Cystip1a absent. Basidia not observed. 
Basipiosporss cylindrical, hyaline, thin-walled, smooth, inamyloid, 7-11 x 
3-4 um. 
EcoLoGy & DIsTRIBUTION: On deciduous wood. According to Ryvarden & Johansen 
(1980), the species is pantropical. It is new to Brazil. 


SPECIMENS EXAMINED: BRAZIL. PaRA, Floresta Nacional de Caxiuana, VIII.2007, leg. 
PS Medeiros et al. PS122, PS226, PS342 (MG195152, MG195155, MG195182). 


REMARKS: A cigar brown to fuscous black pileus surface, rather large shallow 
pores, and broad dextrinoid skeletal hyphae characterize the species. 


Coriolopsis byrsina (Mont.) Ryvarden, Norw. Jl Bot. 19: 230 (1972). 

DEscrIPTION: This species is characterized by small pores, soft rusty-brown 
basidiomata, and ellipsoid to sub-cylindrical basidiospores, 9-14 x 4.5-5 um. 
For further details see Ryvarden & Johansen (1980). 


EcoLoGy & DISTRIBUTION: On deciduous wood. In tropical Africa, America, and Asia 
(Ryvarden & Johansen 1980; Dai 2011). In Brazil, previously known from the states of 
Acre, Mato Grosso, Rio Grande do Sul, and Sao Paulo (Gugliotta & Abrahao 2011); 
newly reported from Amazonas, Ronddénia, and Roraima. 

SPECIMENS EXAMINED: BRAZIL. Acre: Rio Branco, 24.1X.1980, leg. B. Lowy et al. 
241 (INPA 183806); AMAZONAS: Presidente Figueiredo, 18.1.1983, leg. M.A Jesus 123 
(INPA 183806); Maro Grosso: Aripuana, 23.IV.1978, leg. M.A. Sousa 427 (INPA 
75606); RONDONIA: Jaru, Reserva Bioldgica de Jaru, 13.V.1987, leg. M. Capelari et al. 
(SP 211995); location not determined, 4.VII.1968, leg. K.P. Dumont et al. 143 (INPA 
65179); 3.111984, leg. GJ. Samuels 55 (INPA 129095); RorAIMA: locality unknown, 
1.XII.1977, leg. I.J. Aratijo et al. 757 (INPA 78476); SAo PAuLo: Itaicy, 06.VII.1957, leg. 
not determined (URM 7880, as Polystictus byrsinus). 


Coriolopsis caperata (Berk.) Murrill, N. Amer. Fl. 9(2): 77 (1908). 

DESCRIPTION: Coriolopsis caperata is characterized by effused-reflexed to 
pileate darkly colored basidiomata and a zonate abhymenial surface in different 
shades of brown. For further details see Ryvarden & Johansen (1980). 


ECOLoGy & DISTRIBUTION: On deciduous wood. Tropical Africa and America (Ryvarden 
& Johansen 1980). In Brazil, it is reported for the states of Acre, Alagoas, Amapa, 
Bahia, Espirito Santo, Mato Grosso, Minas Gerais, Para, Paraiba, Parana, Pernambuco, 
Rio Grande do Norte, Rond6énia, Roraima, Rio de Janeiro, Rio Grande do Sul, Santa 
Catarina, Sao Paulo, Sergipe (Baltazar & Gibertoni 2009, Gugliotta & Abrahao 2011), 
and now for Amazonas State. 


SPECIMENS EXAMINED: BRAZIL: Acre: Sena Madureira, 27.1X.1968, leg. G. T. Prance 
et al. 7595 (INPA 24394); AMAZONAS: Barcelos, II.1984, leg. G. J. Samuels 72 (INPA 
129121); Borba, 6.V.1985, leg. K.F. Rodrigues et al. 490 (INPA 129022, as Coriolus 
versicolor); Manaus, 1.V.1977, leg. M. A. Souza 149 (INPA 74657, as Coriolopsis polyzona); 
6.X.1985, leg. K.E. Rodrigues et al. 800 (INPA 137086); 5.X.1927, leg. P. Occhioni (SP 
25530); 23.XII.1983, leg. G. Guzman et al. (SP 193577); Manicoré, IV.1985, leg. K.F. 
Rodrigues et al. 91 (INPA 128914, as Coriolopsis sp); Nova Aripuana, 29.IV.1985, leg. 


226 ... Nogueira-Melo & al. 


K.F. Rodrigues et al. 410, 389 (INPA 129009, as C. polyzona, INPA 129005, as Polyporus 
sp.); Tapuruquara, 22.1.1978, leg. I. J. Araujo et al. 973 (INPA 78745, as Hexagonia 
sp.); Tefé, 11. VII.1973, leg. E. Lleras et al. 16611 (INPA 39800); Cachoeira de Taruma, 
5.X.1927, leg. P. Occhioni (RB 217241); Serra de Araga, 29.11.1984, leg. G. J. Samuels 
(RB 24785); Manicoré, Estrada do Estanho, 20.IV.1985, leg. K.F Rodrigues (RB 238166); 
Novo Aripuana, Vila do Apuy, 29.IV.1985, leg. K.F. Rodrigues et al. (RB 238334); Distrito 
Agropecuario da Suframa, 03.X.1985, leg. N. Rodrigues et al. (RB 237012, RB 240587); 
PaRA: Belém, 13.IX.1968, leg. B. Santiago (SP 106707); 3.VI.1980, leg. V. L. Bononi (SP 
177467); Melgaco, VIII.2007, leg. T. B. Gibertoni (URM 79656 on Xylopia sp; URM 
79653 on Pouteria sp.; URM 79650 on Nectandra sp.; URM 79649 on Licania sp.; URM 
79646 on Parkia sp.; URM 79647 on Sclerolobium sp.; URM 79648 on Eschweilera sp.; 
URM 79652 on Dinizia excelsa); Redengao, 20.VIII.1984, leg. N. Rodrigues (RB 224462); 
Oriximina, 28.VI.1980, leg. V.L. R. Bononi et al. 638, 241 (INPA 103609, as Coriolus sp., 
INPA 103335, as Trametes pinsita); Itaituba, [X.1977, leg. M.A. Sousa et al. 25 (INPA 
74631, as Hexagonia caperata); PERNAMBUCO: Escada, 26.X.1954, leg. not determined 
(URM 1010, as Polystictus occidentalis); Recife, 24.V1I.1957, leg. not determined (URM 
12468, as P. caperatus); Tapera, 1932, leg. not determined (URM 762, as P. caperatus); 
RONDONIA: Caracarai, 3.VII.1968, leg. G.T. Prance et al. 5497 (INPA 22257); Campo 
Novo, X.1979, leg. R.H. Petersen 155 (INPA 110517, as Polyporus sp.); Jaru, 2.X.1986, 
leg. M. Capelari & R. Maziero (SP 211273); Vilhena, X.1979, leg. R. H. Petersen 218 
(INPA 110706, as Polyporus sp.); Porto Velho, Estacao Ecoldgica de Cunia, I1.2007, leg. 
A. C. Gomes-Silva 33 (URM 78898); II.2008, leg. A. C. Gomes-Silva 222 (URM 78906); 
11.11.2010, leg. A.C. Gomes-Silva et al. 815 (URM 82837); Parque Natural Municipal 
de Porto Velho, VII.2007, leg. A. C. Gomes-Silva 59 (URM 78900); VII.2008, leg. A.C. 
Gomes-Silva 603 (URM 79552); 20.V1.2009, leg. A.C. Gomes-Silva et al. 738 (URM 
82833); 29.1.2010, leg. A.C. Gomes-Silva et al. 941 (URM 82831); 3.11.2011, leg. A.C. 
Gomes-Silva et al. 1089 (URM 82903); Fazenda Mucuim, VII.2007, leg. A.C. Gomes- 
Silva 91 (URM 78901); RoraimMa: Alto Alegre, 21.V1.1986, leg. K.E Rodrigues et al. 
1083 (INPA 143425); Caracarai, 16.X1.1977, leg. I .J. Araujo et al. 438 (INPA 76933, as 
Hexagona sp.). 


Coriolopsis floccosa (Jungh.) Ryvarden, Norw. Jl Bot. 19(3-4): 230 (1972). 

DESCRIPTION: The umber brown tomentose to hirsute or even velutine 
pileus surface and fairly large pores are diagnostic for this species. For further 
details see Ryvarden & Johansen (1980). 


EcoLoGy & DISTRIBUTION: On deciduous wood. Pantropical (Ryvarden & Johansen 
1980). In Brazil, reported for the states of Alagoas, Bahia, Para, Paraiba, Parana, 
Pernambuco, Rio Grande do Sul, Rondénia, Roraima, Santa Catarina and Sao Paulo 
(Gugliotta & Abrahao 2011). Newly recorded for Acre, Amazonas, and Mato Grosso. 


SPECIMENS EXAMINED: BRAZIL: Acre: location not determined, 3.X.1980, leg. B. 
Lowy et al. 1069 (INPA 100886, as Polyporus sp.); AMAZONAS: Manaus, 10.X.1989, 
leg. R. Carvalho 1332 (INPA 192503, as Coriolopsis sp.); 21.V1.1985, leg. M. A. Jesus 
755 (INPA 185984); 10.[X.1977, leg. M. A. Sousa & I. J. Aratjo 219 (INPA 74661, as 
Coriolus maximus); Itacoatiara, 21.XII.1966, leg. G. T. Prance et al. 3778 (INPA 19403, 
as Coriolopsis sp.); 10.1X.1980, leg. B. Lowy et al. 129 (INPA 100040, as Polyporus sp.); 
Bauia: Conde, 08.11.1955, leg. A.C. Batista (URM 1242, as Polystictus pinsitus); Amaro, 
23.V.1957, leg. not determined (URM 7450, as Polystictus membranaceus); MATo 


Coriolopsis in Brazil ... 227 


Grosso: Aripuana, 23.IV.1978, leg. M.A. Sousa et al. 431 (INPA 75610); PARA: Belém, 
17.1V.1979, leg. I.J. Araujo 1239 (INPA 102851); Itaituba, 3.X.1977, leg. M.A. Sousa et 
al. 140 (INPA 74538, as Coriolopsis sp.); PERNAMBUCO: Recife, 23.VII.1955, leg. not 
determined (URM 12498, as P. membranaceus); RIO GRANDE DO SUL: Sao Leopoldo, 
1932, leg. J. Rick (URM 9023, as Polystictus rigens); RORAIMA: Alto Alegre, 16.V1.1986, 
leg. E.S.S. Silva et al. 493 (INPA 154958); Boa Vista, 19.VII.1989, leg. M.A. Jesus 880 
(INPA 186185, as Coriolopsis sp.); 1.X1I.1977, leg. L.L.J. Aguiar et al. 750 (INPA 78469, 
as Coriolopsis sp.); 31.1.1984, leg. G.J. Samuels 39 (INPA 129089); RONDONIA: Porto 
Velho, Estacao Ecolégica de Cunia, VII.2007, leg. A.C. Gomes-Silva 204 (URM 78909); 
Parque Natural Municipal de Porto Velho, VII.2007, leg. A.C. Gomes-Silva 186 (URM 
79477, as Coriolopsis rigida); Fazenda Mucuim, VII.2007, leg. A.C. Gomes-Silva 258 
(URM 79478, as C. rigida). 


Coriolopsis hostmannii (Berk.) Ryvarden, Syn. Fung. 23: 39 (2007). 

BASIDIOMATA perennial, sessile, hard and brittle when dry, dimidiate to 
flabelliform, solitary, projecting 2-4 cm, 3-5 cm wide and 0.5-1.5 mm thick. 
ABHYMENIAL SURFACE glabrous, dull to subshiny, zonate with brown and purple 
zones, hazel (27) to drab (33). MARGIN acute, concolorous with abhymenial 
surface. CONTEXT homogeneous, < 0.5 mm thick, fulvous (12) to cigar brown 
(16). Tubes concolorous with context, < 0.5 mm long. HYMENIAL SURFACE with 
pores round, 6-7 per mm, dissepiments 50-100 um thick, cigar brown (16). 
HYPHAL SYSTEM trimitic, generative hyphae hyaline, clamped, thin-walled, 
1.5-2.5 um diam.; skeletal hyphae yellowish to pale brown, thick-walled, 4-7.5 
um diam.; binding hyphae golden-yellow, almost solid, 3-5 um diam. CysTIDIA 
absent. BAsIp1A not observed. Bastp1osPorEs cylindrical, hyaline, thin-walled, 
inamyloid, 8-10 x 2.5-4.0 um. 

EcoLoGy & DISTRIBUTION: On deciduous wood. Neotropical, from Southern United 
States and south to Venezuela (Ryvarden & Johansen 1980). It is new to Brazil. 


SPECIMENS EXAMINED: BRAZIL. PERNAMBUCO: Paulista, Manguezal de Maria Farinha, 
on dead Rhizophora mangle, 20.VII.2009, leg. G.S. Nogueira-Melo et al. NM003 (URM 
82147); on dead Avicennia schaueriana, 31.1.2010, leg. G.S. Nogueira-Melo et al. NM002 
(URM 82146); Itamaraca, Manguezal do Canal de Santa Cruz, on live A. schaueriana, 
25.V1.2009, leg. G. S. Nogueira-Melo et al. NM002, NM025 (URM 82134, URM 82138); 
on dead A. schaueriana, 10.III.2009, leg. G.S. Nogueira-Melo et al. NM001, NM004 
(URM 82136, 82145); 26.VI.2009, leg. G.S. Nogueira-Melo et al. NM003, NMO07, 
NMO011 (URM 82144, URM 82143, 82137); 26.VII.2009, leg. G.S. Nogueira-Melo et 
al. NM002 (URM 82139); 22. VIII.2009, leg. G.S. Nogueira-Melo et al. NM006, NM012 
(URM 82141, URM 82140); 17.1.2010, leg. G.S. Nogueira-Melo et al. NM002 (URM 
82135); 19.1I1.2010, leg. G.S. Nogueira-Melo et al. NM004 (URM 82142). 


REMARKS: Coriolopsis hostmannii may be confused with C. aspera, which has 
similar hyphae and basidiospores. However, C. hostmannii has smaller pores 
(3-4 pores per mm in C. aspera) and smooth pilei. The zonate abhymenial 
surface may cause the species to be mistaken for Funalia polyzona (Pers.) 
Niemela, although Funalia usually has a distinct variably zoned tomentum. 


228 ... Nogueira-Melo & al. 


Coriolopsis psila (Lloyd) Ryvarden, comb nov. 
MycosBank MB561160 
= Fomes psila Lloyd, Syn. Fomes: 233 (1915). [BPI!]. 
= Phylloporia psila (Lloyd) Ryvarden, Norw. Jl Bot. 19: 235 (1972). 

BASIDIOMATA annual to perennial, sessile, solitary, sessile, pileus dimidiate 
to flabelliform, < 6 cm in diam. and 2 cm thick with the tomentum, the latter 
as a dense mat of intertwined hairs in reddish brown colours, < 1 cm thick. 
CONTEXT cigar brown (16) to umber (18), < 4 mm thick in lower denser part, 
no black zone towards the cottony dense hair surface mat, tubes single layer 
or stratified as in the type, individual layer 3-4 mm thick. HYMENIAL SURFACE 
rusty-brown; dissepiments entire, relatively thick; pores round, 6-7 per mm, 
almost invisible to the naked eye. HYPHAL SYSTEM trimitic, generative hyphae 
often collapsed and difficult to observe, hyaline, thin-walled, but always with a 
distinct lumen often with secondary adventitious septa, < 8 um in diam., binding 
hyphae irregular in outline, frequently branched or with a few long tapering 
branches, thick-walled, yellow to light brown, < 6 um in diam. BASIDIOSPORES 
cylindrical, 9-12 x 3-4.5 um, hyaline, thin-walled and non-amyloid. 

EcoLocy & DISTRIBUTION: On deciduous wood. Known from Brazil and Mexico. The 
type locality in Brazil is not detailed but J. Rick, who collected the material, used to work 


in Rio Grande do Sul State, South Brazil. If so, it is probably a new occurrence for the 
Brazilian Amazonia and Northeast Brazil. 


SPECIMENS EXAMINED: BRAZIL. AMAzoNAS: Manaus, 20.IV.1985, leg. M.A. Jesus 
709 (INPA 185947, as Perenniporia sp.); 2.V.1990, leg. M.A. Jesus 1384 ([NPA 192652, 
as Microporus sp.); Manicoré, 20.IV.1985, leg. K.E Rodrigues 233 (INPA 128955, as 
Polyporus sp.); BAHIA: location not determined, date not determined, leg. C. Torrend 
(O 10486); Gongogi, V.1910, leg. C. Torrend, s.n. (URM 8893, as Trametes ocellata Berk. 
& M.A. Curtis); RONDONIA: location not determined, 21.[X.1980, leg. B. Lowy et al. 230 
(INPA 185947, as Perenniporia sp.); 20.V.1987, leg. M. Capelari et al. (SP 212021). 
EXTRALIMITAL SPECIMEN EXAMINED: MEXICO. Oaxaca: Tehuantepec, 13.V1.1976, 
leg. A.L. Welden 3606 (O 10488). 
REMARKS: Coriolopsis psila is a conspicuous species due the dense thick mat 
of entangled reddish brown hairs covering the pileus. Superficially it reminds 
one of H. hydnoides, which, however, has black and stiffer hairs on the pileus. 
Basidiospores of the two species are similarly cylindrical, but those of H. 
hydnoides are longer (12-14 um). The generic concepts among polypores 
with a trimitic hyphal system producing white rot (Coriolopsis, Hexagonia, 
Pycnoporus, Trametes) are unclear and are based in part on basidioma color, a 
character of dubious value at the generic level. Further DNA sequencing will 
reveal the true phylogeny within these genera. For the time being we place F. 
psila in Coriolopsis due to its brown context and a spore length that is closer to 
other Coriolopsis species than in Hexagonia. 
The combination in Phylloporia was based on the presence of a black line 
between the hairy cover of the pileus and context, a prominent character for 


Coriolopsis in Brazil ... 229 


Phylloporia species. However Phylloporia was later characterized by small 
ellipsoid basidiospores and a parasitic life strategy (Wagner & Ryvarden 2002), 
while C. psila is saprophytic and has larger basidiospores. Seemingly a biological 
convergent character, the hairy pilear covering occurs in many polypore genera 
and is apparently an adaption to resist drying. 


Key to the species of Coriolopsis recorded in Brazil 


la/Basidiomata mostlysessile:. ir... sa. <s awe haa le 2 hale a Oka + Mao os uae Oe ee OS 2 
1b Basidiomata-mostly-efhused=retlexed iia dys ssi-s dis steadringcion dine drs Pedy s ea dared 5 
Das Weices~ led spel alii titer Bet. weet art ea aes Game! aed eet baRMags hoRteks (ecient Lacie ball 3 
2D. ORES O= 7? DE MIE: ef DFImGd AsRGd As RG EM st ME MOR Alder UR Alden carer Pedces 4 
3a. Abhymenial surface finely tomentose at the margin, pores angular, 

1-2 per mm, basidiospores not known ................ ee eee eeee C. burchellii 
3b. Abhymenial surface with scrupose tuft of agglutinated hairs, pores round, 

3-4 per mm, basidiospores cylindrical 9-16 x 3-5 um .............. C. aspera 
4a, Abhymenial surface glabrous, basidiospores 8-10 x 2.5-4 um ....... C. hostmannii 
4b. Abhymenial surface with a dense mat of intertwined hairs, 

basidiospores 9-12 GAs 3 se,-une, ste ines ie tuna in en nue tee C. psila 
5a. Basidiospores oblong ellipsoid to subcylindrical, 10-14 x 4.5-6 um, 

hyaline. tosvery; palesellowiShiss.tccerm vtscrem, Fst, wala rites tarn ities eral on dg C. byrsina 
5b. Basidiospores ellipsoid to cylindrical, fusiform, hyaline, 7-11(-14) x 2-4 um..... 6 
6a. Context distinctly duplex, tobacco-brown and shiny................... C. floccosa 
6b. Context mostly homogeneous, dark brown to bay and dull..................... 7 


7a. Context distinctly darker than the tubes, hymenial surface bay to deep brown, 
pores round to angular, (2-)4-5 permm..................00. C. brunneoleuca 


7b. Context dark date-brown concolorous with tubes, hymenial surface ochraceous, 
to cinnamon to deep chocolate brown, pores round, 3-5 permm ....C. caperata 


Acknowledgments 

We would like to thank Dr. YC Dai and Dr. P. Buchanan for critically reviewing this 
manuscript; Ana Cristina R. Souza, curator of the HFSL, for support during the field 
trips of ACGS; Carlos Franciscon, curator of INPA and Adriana Gugliotta, curator of SP, 
for the loan of exsiccates; Anibal Alves de Carvalho Junior for support during the visit 
to RB; the staff of the ECFPn and of the MPEG for support during the field trips of PSM 
and TBG; and the Programa de Pesquisa em Biodiversidade (PPBio) da Amazonia for 
support during the field trips of PSM. Further, we acknowledge the Conselho Nacional 
de Desenvolvimento Cientifico (CNPq) for the master scholarship of ACGS, GSNM and 
PSM; the Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (Capes) for 
the doctorate scholarship of ACGS; the Instituto Internacional de Educagao do Brasil 


230 ... Nogueira-Melo & al. 


(IEB) and the Gordon and Betty Moore Foundation for the Scholarship of Studies on 
Amazonia Conservation (BECA) to ACGS, PSM and TBG; the Dottorato di Ricerca in 
Ecologia Sperimentale e Geobotanica (Universita degli Studi di Pavia, Italy), the Pés- 
Graduacao em Biologia de Fungos (UFPE, Brazil), the Instituto Nacional de Ciéncia e 
Tecnologia - Herbario Virtual de Plantas e Fungos (CNPQ 573883/2008-4) and FACEPE 
(APQ 0433-2.03/08) for partially financing this study. 


Literature cited 

Baltazar JM, Gibertoni TB. 2009. A checklist of the aphyllophoroid fungi (Basidiomycota) recorded 
from the Brazilian Atlantic Forest. Mycotaxon 109: 439-422. 
http://dx.doi.org/10.5248/109.439 

Bononi VLR. 1984. Basidiomicetos do Cerrado da Reserva Biolégica de Moji-Iuacu, SP.Rickia 11: 
1-25. 

Dai YC. 2012. Polypore diversity in China with an annotated checklist of Chinese polypores. 
Mycoscience 53: 49-80. http://dx.doi.org/10.1007/s10267-011-0134-3 

Dai YC, Cui BK, Yuan HS, He SH, Wei YL, Qin WM, Zhou LW, Li HJ. 2011. Wood-inhabiting fungi 
in southern China 4. Polypores from Hainan Province. Ann. Bot. Fennici 48: 219-231 

Gibertoni TB, Ryvarden L, Cavalcanti, MAQ. 2004. Poroid fungi (Basidiomycota) of the Atlantic 
Rain Forest in Northeast Brazil. Syn. Fung.18: 33-43. 

Gilbertson RL, Ryvarden L. 1986. North American polypores 1. Fungiflora, Oslo. 

Kirk PM, Cannon PF, Minter DW, Stalpers JA. 2008. Dictionary of the Fungi. 10 ed. CABI 
Publishing, Surrey. 771 p. 

Nufiez M, Ryvarden L. 2001. East Asian polypores 2. Polyporaceae s. lato. Synopsis Fungorum 14: 
170-522, 

Ryvarden L. 1991. Genera of Polypores - Nomenclature and taxonomy. Synopsis Fungorum 5: 
1-363. 

Ryvarden L, Gilbertson RL. 1993. European Polypores, part 2. Synopsis Fungorum 7: 394-743. 

Ryvarden L, Johansen I. 1980. A preliminary polypore flora of East Africa. Oslo, Fungiflora, 630 p. 

Tavares I. 1939. Catalogo dos fungos de Pernambuco. Bol. Secr. Agric. (Pernambuco) 4(1): 1-33. 

Wagner T & Ryvarden L. 2002: Phylogeny and taxonomy of the genus Phylloporia (Hymenochaetales). 
Mycol Progress 1:105-116. http://dx.doi.org/10.1007/s11557-006-0009-8 

Wang B, Cui BK, Li HJ, Du P, Jia BS. 2011. Wood-rotting fungi in eastern China 5. Polypore 
diversity in Jiangxi Province. Ann. Bot. Fennici 48: 237-246. 


ISSN (print) 0093-4666 © 2012. Mycotaxon, Ltd. ISSN (online) 2154-8889 


MY COTAXON 


http://dx.doi.org/10.5248/120.231 
Volume 120, pp. 231-237 April-June 2012 


A new species of Postia (Basidiomycota) from Northeast China 


Bao-Kai Cur & Hat-Jiao Li 


Institute of Microbiology, P.O. Box 61, Beijing Forestry University, 
Beijing 100083, China 
*CORRESPONDENCE TO: baokaicui@yahoo.com.cn 


ABSTRACT — A new polypore, Postia subplacenta sp. nov. from Northeast China, is described 
and illustrated. It is characterized by resupinate basidiocarps, angular pores (3-5 per mm) 
with a cream buff to pale cinnamon-buff pore surface, and cylindrical basidiospores (4.2-6.0 
x 1.9-2.4 um) that are negative in Melzer’s reagent and Cotton Blue. A key to accepted species 
of Chinese Postia is supplied. 


Key worps — lignicolous, poroid, fungi, Fomitopsidaceae, taxonomy 


Introduction 

Postia Fr. (Polyporales, Basidiomycota) is an important genus of brown-rot 
fungi that are widely distributed in the northern hemisphere and mostly grow 
on gymnosperm wood. Some species of the genus are economically important, 
such as Postia guttulata (Peck) Jiilich and P lactea (Fr.) P. Karst., which have 
been used as medicine in China (Dai et al. 2009a). 

Some mycologists consider Postia a synonym of Oligoporus Bref. (Gilbertson 
& Ryvarden 1987, Nufiez & Ryvarden 2001, Ryvarden & Gilbertson 1994), 
while others regard it as an independent genus (Dai & Hattori 2007, Larsen 
& Lombard 1986, Niemela et al. 2004, Renvall 1992, Wei & Dai 2006). The 
genus is characterized by an annual growth habit, a monomitic hyphal system 
with clamp connections, and thin-walled, allantoid to cylindrical or ellipsoid 
basidiospores. Twenty-eight species have previously been known from China 
(Cui et al. 2008, Dai 2009, Dai et al., 2003, 2004, 2007a,b, 2009b, Dai & Penttila 
2006, Li et al. 2007, 2008, Wang et al. 2009, 2011, Wei & Dai 2006, Wei & Qin 
2010, Yuan & Dai 2008, Yuan et al. 2010). 

During a study on the taxonomy and diversity of wood-rotting fungi in 
Northeast China, two Postia specimens were found that represented one 
previously undescribed species, which we propose as P. subplacenta. In addition, 
we provide an identification key to the Postia species thus far reported from 
China. 


2352-,.. Cui & Li 


Materials & methods 

The studied specimens were deposited in the herbarium of Beijing Forestry University 
(BJFC). The microscopic procedure follows Dai (2010) and Cui et al. (2011). In 
presenting the variation in the size of the spores, we exclude 5% of measurements (given 
in parentheses) from each end of the range. The following abbreviations are used: IKI = 
Melzer’s reagent, IKI- = negative in Melzer’s reagent, KOH = 5% potassium hydroxide, 
CB = Cotton Blue, CB- = acyanophilous, L = mean spore length (arithmetic average of 
all spores), W = mean spore width (arithmetic average of all spores), Q = variation in 
the L/W ratios between the specimens studied, n = number of spores measured from 
given number of specimens. Sections were studied at magnifications up to x1000 using 
a Nikon Eclipse E 80i microscope and phase contrast illumination. Drawings were made 
with the aid of a drawing tube. Special colour terms follow Petersen (1996). 


Taxonomy 


Postia subplacenta B.K. Cui, sp. nov. FIG. 1 
MycoBank MB 563784 


Differs from Postia placenta in the cream-buff to pale cinnamon-buff pore surface, wider 
subicular hyphae, smaller basidiospores, and absence of gloeopleurous hyphae. 


Type. — China. Jilin Province, Antu County, Changbaishan Nature Reserve, on fallen 
trunk of Pinus, 8.VII.2011, Cui 10001 (holotype, BJFC). 


EryMoLocy — subplacenta (Lat.) denotes a similarity with Postia placenta. 

FRuITBODY — Basidiocarps annual, resupinate, corky when fresh, corky to 
fragile when dry, up to 5 cm long, 3 cm wide, and 2.5 mm thick at the center. 
Pore surface white to cream-buff when fresh, cream-buff to pale cinnamon- 
buff when dry; pores angular, 3-5 per mm; dissepiments thin, entire to lacerate. 
Margin indistinct, narrow to almost lacking. Subiculum cream to cream-buff, 
corky when dry, <0.3 mm thick. Tubes paler than pore surface, pale cream-buff, 
fragile when dry, <2.2 mm long. 

HYyPHAL STRUCTURE — Hyphal system monomitic; generative hyphae 
bearing clamp connections, IKI-, CB-; tissues unchanged in KOH. 

SUBICULUM — Generative hyphae hyaline, thin- to mostly slightly thick- 
walled, occasionally branched, interwoven, 3-7 um in diam. 

TuBes — Generative hyphae hyaline, slightly thick- to distinctly thick- 
walled, with a wide to narrow lumen, occasionally branched, interwoven, 2-5 
tum in diam. Cystidia and cystidioles absent. Basidia clavate with four sterigmata 
and a basal clamp connection, 12-18 x 4-5 um; basidioles in shape similar to 
basidia, but slightly smaller. 

Spores — Basidiospores cylindrical, hyaline, thin-walled, smooth, IKI-, 
CB-, (4.0-)4.2-6.0(-6.9) x 1.9-2.4(-2.5) um, L = 5.07 um, W = 2.11 um, 
Q = 2.37-2.45 (n = 60/2). 

TYPE OF ROT — Brown rot. 


ADDITIONAL SPECIMEN EXAMINED — CHINA. HEILONGJIANG PROVINCE, YICHUN, 
Fenglin Nature Reserve, on stump of Pinus, 1. VIII.2011 Cui 9818 (BJFC). 


Postia subplacenta sp. nov. (China) ... 233 


10 ym 


Fic. 1. Postia subplacenta (holotype), microscopic structures. 
a: Basidiospores. b: Basidia and basidioles. c: Hyphae from trama. d: Hyphae from subiculum. 


REMARKS — Postia subplacenta resembles P. placenta (Fr.) M.J. Larsen & Lombard 
in having resupinate basidiocarps, similar pores, and cylindrical basidiospores; 
however, P. placenta has a salmon-pink pore surface, gloeopleurous hyphae, 
narrow subicular hyphae, and larger basidiospores (5.5-7 x 2-2.5 um; 
Gilbertson & Ryvarden 1987, Ryvarden & Gilbertson 1994). 

Postia subplacenta is similar to P. rancida (Bres.) M.J. Larsen & Lombard, 
which differs in having rancid taste and larger basidiospores (6-8 x 2-3 um; 
Gilbertson & Ryvarden 1987). 

Postia obliqua Y.L. Wei & W.M. Qin, recently described from Southwest 
China (Wei & Qin 2010), may be confused with P subplacenta in its resupinate 


234 ... Cui & Li 


basidiocarps and similar pores and basidiospores (4.8-6.3 x 2.0-2.5 um) 
but differs in its very large basidiocarps with oblique tubes that usually form 
imbricate pseudopilei and the presence of gloeopleurous-like hyphae. 


Macroscopically, Postia subplacenta looks somewhat like Oligoporus rennyi 


(Berk. & Broome) Donk, which, however, has chlamydospores and produces 
basidiospores that are oblong-ellipsoid and thin- to slightly thick-walled 
(Ryvarden & Gilbertson 1994). 


Key to species of Postia in China 


1 
1 
2 
2 
3 
3 
4 
4 
S 
Di 
6 
6 
z 
A 
8 
8 
2) 
9 


. Basidiocarps effused-reflexed, pileate or stipitate ............ 0. . eee eee eee 2 
J BasidiGcarps Tesupinates, ows v6 Gea eh oes GS eG ae BGR a eG Ree SG Sx ea eBe Lage ta 23 
, Basidiocarps stipitate or substipitate::.. «<i. + saihece a dinar a diner theta heeees aes 3 
. Basidiocarps effused-reflexed or pileate ..... cece eee eee eens 4 
SUPOTES PL SHPST MIMD 5.25.5}. 4. cgtiabea chelated ties loa atte P. subundosa Y.L. Wei & Y.C. Dai 
Pores: 3=5:per mii. |... 24). 4g hide gale ea dine P. ceriflua (Berk. & M.A. Curtis) Jiilich 
. Basidiocarps with distinct grey to bluish tints ............ 0... cece eee eee eee 2 
. Basidiocarps white, cream, yellowish or brown ............ 0.00. c cece eee eee eee 7 
AD IIEATY STO SO CLIN an chew Rho Rae ha le, ere Mend tN P. alni Niemela & Vampola 
Omevinnospenny, Wo Sa, ha, Moh, Rab, eoie » okie emis Wuohicy ations wabanhery a tieate. att 6 
. Basidiospores; <1.8-wm wide 2.0... 2.00 oes ow Pel ae Jace oe P. caesia (Schrad.) P. Karst. 
. Basidiospores >1.8 um wide ................20005 P. luteocaesia (A. David) Julich 
. Basidiocarps becoming brown when bruised or when dry ..................0.. 8 
. Basidiocarps unchanged when bruised or dry ............ 0.0 cece eee eee eee ee 10 
Molo acyStidia PRESENt «oc p ions eek bance thee ees P. leucomallella (Murrill) Jiilich 
. GOSOCYSUIC IAD SEN tse fap. cwsse don conse tor asosce dan. seraplat Sure gl atsse, pebin:dnsraal ates deh deu ed eaeh Tucows 9 
. Basidiosporesr<ls6 pmwide 062.60 eon Soha eect hee the P lateritia Renvall 
Basidigspores:2 9-7 Unik Wide! oo aoa skate meee meee eee 2 P. fragilis (Fr.) Jalich 
. Basidiocarps chalky when dry ................... P. calcarea Y.L. Wei & Y.C. Dai 
. Basidiocarps fragile, corky or woody hard when dry ...............0....0005 11 
HCY STIAIT PECSONE YS cn elit. darehs teats naa ale teats tmkhs nants toma hs baer ban aie 12 
MEV StH IA <A DG CIE ia dlrs dhe cies fy Fovgion dy tenes dus poten Fy pate as pckerg Pyle chew byt peckow snyas php sdyht gE 14 
Gv sticlia thin=wealled ; fu, siete. totes mekbey Soke 4 P. amurensis Y.C. Dai & Penttila 
PE yotid ia thick swale: ae. o actus spe tarace teanache deavache tawnche tearaghadearaces ae Okee 13 
. Cystidia amyloid in Melzer’s reagent ..... P. pileata (Parmasto) Y.C. Dai & Renvall 
. Cystidia inamyloid in Melzer’s reagent ................ P. balsamea (Peck) Jiilich 
POLES DIAOeT Os POE TINS (ies ie eae wg a ad gd dg P. undosa (Peck) Jiilich 
Rovesssinaller; > 3. periinis 4.07 Pit.) Rist o8! Wn! Wah PN Ae WW tam D Bel! oe 15 


15. 
15. 
16. 
16. 
Le 
17. 
18. 
18. 
eee 
19. 
20. 
20. 


2 
2 


—_—_ —_ 


22; 
22 
23. 
23. 
24. 
24, 
25: 
25: 
26. 
26; 
2a 
27, 
28. 
Ze: 
205 
2S, 


Postia subplacenta sp. nov. (China) ... 235 


Gloeocystidiapresent:..6 2. ces 2. lek Rsk, Re. FENG. SEE on SER EBS i RS cn ES 16 
COCOGVSEIAT AA DSER so 35 cect crease ore i eee tere Ee en een eed Nera, ae 17 
Hyphal pegs abundant .................... P. gloeocystidiata Y.L. Wei & Y.C. Dai 
Hyphal-pegs.absetit. (e520 Gin 2.506.250 28 since a P. qinensis Y.C. Dai & Y.L. Wei 
Piléal surface more. or less: prvkewhencfréshicy, soacts seats seacts soadts seats o oats: 18 
Pileal surface-never putk when: fteshi ag 40 ack 9 esp cto Het acdeg Sh poe Shp toe Fy pee gasp al 19 
Basidiospores sds57pim- wide 1.8. i. weds wees 8 P. persicina Niemela & Y.C. Dai 
Basidiospores: 15: (it Wide" w.s00,"t-caccts tearacts saracte dive P. cana H.S. Yuan & Y.C. Dai 
Pilealtstikface-7Onate<w 28 pot.2o Pito8 Bite whet tat 08s P. zebra Y.L. Wei & Y.C. Dai 
Pileall SUPEACE AZ ON ALC op tartes Biemer thy Besma tin Meaney Wemed ee Beaman Mendel NeLuadd nahtonade wiktada beth 20 
Basidiocarps mild, upper surface greyish brown........ P. tephroleuca (Fr.) Jitlich 
Basidiocarps bitter, upper surface cream or yellow-brownish ................. 21 

b, BASTCAOSPOTES! 32 SNL WIE” s0s-s0- notary pots wild Ges atlas Sed cwie ota ve Gte sae a BS P. guttulata 

FAIS AST IOS PONG SES ce MUTA OVI cs ic PR ol Ud Baad Sad ta a LOGI ad alr ca dete heen 22, 
Basidiocarps woody hard when dry .................06. P. stiptica (Pers.) Julich 
Basidiocarps-trasileswhetirdry Sasss0-e5tass aoe ie Siluueesta we oed ale Ba P. lactea 
Basidiocarps becoming reddish to rusty brown when touched......... P lateritia 
Basidiocarpsunchanged when touched 2.64 .-..csge i tage v atetage Be ge Hee lage Bley 24 
Basidiospores mostly >2 um wide, cystidia absent ................... 0.0 25 
Basidiospores mostly <2 um wide, cystidia present .................. 00000 28 
Basidiocarps salmon pink; basidiospores 2-3 um wide ............... P. placenta 
Basidiocarps white or cream; basidiospores 2-2.5 um wide ...............4-. 26 
Basidiocarps suitti-ranicid-srielll, ch. sey tho isem adler whe My ele ake ghtwsbie P. rancida 
Basidiocarps wati@ut, PAN CTS Stivell sys, 1dr Heer Diese ace anda dae Seas tae ee aero oe 27 
Basidiocarps very large, gloeopleurous hyphae present ................ P. obliqua 
Basidiocarps small, gloeopleurous hyphae absent ................. P. subplacenta 
Cystidia thick-walled spa 52s cic Papa ese et oR gt PR neh lo clo gh ta io P. pileata 
Gysticia- thin: walledee i. 0 ten FOUN ie FE Aes wR nue eee A MRE WSS ee ne, AEe ha, 29 
Pores 5-6 per mm; basidiospores mostly <1 um wide ..... P. simanii (Pilat) Jiilich 
Pores 3-4 per mm; basidiospores mostly >1 um wide 


pie Reet eee Oe ON MEN ote CONE ole toh? P. hibernica (Berk. & Broome) Jiilich 


Acknowledgements 


Special thanks are due to Mr. Chang-Lin Zhao (China) for help in field collection. 


We express our gratitude to Drs. Michal TomSovsky (Czech Republic) and Yu-Lian 
Wei (China) who reviewed the manuscript. The research was financed by the National 
Natural Science Foundation of China (Project No. 31170018) and Program for New 
Century Excellent Talents in University (NCET-11-0585). 


236 ... Cui & Li 


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Mountains, Fujian Province. Sydowia 60: 25-40. 

Cui BK, Li HJ, Dai YC. 2011. Wood-rotting fungi in eastern China 6. Two new species of 
Antrodia (Basidiomycota) from Mt. Huangshan, Anhui Province. Mycotaxon 116: 13-20. 
http://dx.doi.org/10.5248/116.13 

Dai YC. 2009. A checklist of polypores in China. Mycosystema 28: 315-327. 

Dai YC. 2010. Hymenochaetaceae (Basidiomycota) in China. Fungal Diversity 45: 131-343. 
http://dx.doi.org/10.1007/s13225-010-0066-9 

Dai YC, Hattori T. 2007. Postia japonica (Basidiomycota), a new polypore from Japan. Mycotaxon 
102: 113-118. 

Dai YC, Penttila R. 2006. Polypore diversity of Fenglin Nature Reserve, northeastern China. 
Annales Botanici Fennici 43: 81-96. 

Dai YC, Harkénen M, Niemela T. 2003. Wood-inhabiting fungi in southern China 1. Polypores 
from Hunan Province. Annales Botanici Fennici 40: 381-393. 

Dai YC, Wei YL, Wang Z. 2004. Wood-inhabiting fungi in southern China 2. Polypores from 
Sichuan Province. Annales Botanici Fennici 41: 319-329. 

Dai YC, Cui BK, Huang MY. 2007a. Polypores from eastern Inner Mongolia, northeastern China. 
Nova Hedwigia 84: 513-520. http://dx.doi.org/10.1127/0029-5035/2007/0084-0513 

Dai YC, Cui BK, Yuan HS. 2007b. Notes on polypores from Gansu and Qinghai Province, northwest 
China. Cryptogamie Mycologie 28: 177-187. 

Dai YC, Yang ZL, Cui BK, Yu CJ, Zhou LW. 2009a. Species diversity and utilization of medicinal 
mushrooms and fungi in China (Review). International Journal of Medicinal Mushrooms 11: 
287-302. http://dx.doi.org/10.1615/IntJ MedMushr.v11.i3.80 

Dai YC, Yuan HS, Wang HC, Yang F, Wei YL. 2009b. Polypores (Basidiomycota) from Qin Mts. in 
Shaanxi Province, central China. Annales Botanici Fennici 46: 54-61. 

Gilbertson RL, Ryvarden L. 1987. North American polypores 2. Fungiflora, Oslo. 434-885 pp. 

Larsen MJ, Lombard FE. 1986. New combinations in the genus Postia Fr. (Polyporaceae). Mycotaxon. 
26: 27-1)-273; 

LiJ, Xiong HX, Zhou XS, Dai YC. 2007. Polypores (Basidiomycetes) from Henan Province in central 
China. Sydowia 59: 125-137. 

Li J, Xiong HX, Dai YC. 2008. Polypores from Shennongjia Nature Reserve in Hubei Province, 
Central China. Cryptogamie Mycologie 29: 267-277. 

Niemela T, Dai YC, Kinnunen J, Schigel DS. 2004. New and in North Europe rare polypore species 
(Basidiomycota) with annual, monomitic basidiocarps. Karstenia 44: 67-77. 

Nujiez M, Ryvarden L. 2001. East Asian polypores 2. Polyporaceae s. lato. Synopsis Fungorum 14: 
165-522. 

Petersen JH. 1996. Farvekort. The Danish Mycological Society’s colour-chart. Foreningen til 
Svampekundskabens Fremme, Greve. 6 p. 

Renvall P. 1992. Basidiomycetes at the timberline in Lapland 4. Postia lateritia n. sp. and its rust- 
coloured relatives. Karstenia 32: 43-60. 

Ryvarden L, Gilbertson RL. 1994. European polypores 2. Synopsis Fungorum 7: 394-743. 

Wang B, Dai YC, Cui BK, Du P, Li HJ. 2009. Wood-rotting fungi in eastern China 4. Polypores from 
Dagang Mountains, Jiangxi Province. Cryptogamie Mycologie 30: 233-241. 

Wang B, Cui BK, Li HJ, Du P, Jia BS. 2011. Wood-rotting fungi in eastern China 5. Polypore 
diversity in Jiangxi Province. Annales Botanici Fennici 48: 237-246. 


Postia subplacenta sp. nov. (China) ... 237 


Wei YL, Dai YC. 2006. Three new species of Postia (Aphyllophorales, Basidiomycota) from China. 
Fungal Diversity 23: 391-402. 

Wei YL, Qin WM. 2010. Two new species of Postia from China. Sydowia 62: 165-170. 

Yuan HS, Dai YC. 2008. Polypores from northern and central Yunnan Province, southwestern 
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http://dx.doi.org/10.1111/j.1756-1051.2010.00849.x 


ISSN (print) 0093-4666 © 2012. Mycotaxon, Ltd. ISSN (online) 2154-8889 


MY COTAXON 


http://dx.doi.org/10.5248/120.239 
Volume 120, pp. 239-246 April-June 2012 


Three new Caeoma species on Rosa spp. from Pakistan 


N.S. AFSHAN”™, A.N. KHALID? & A.R. NIAZI’ 


™Centre for Undergraduate Studies & *Department of Botany, University of the Punjab, 
Quaid-e-Azam Campus, Lahore, 54590, Pakistan 


*CORRESPONDENCE TO: pakrust@gmail.com 


ABSTRACT — Three representatives of the anamorphic genus Caeoma —C. ahmadii on Rosa 
microphylla; C. khanspurense and C. rosicola on Rosa webbiana— are described as new rust 
species from Pakistan. This first report of Caeoma raises the number of known anamorphic 
rust genera from the country to five. 


Key worps — Khanspur, Mansehra, Phragmidium 


Introduction 

The genus Caeoma Link is traditionally used for species having sori that 
lack obvious bounding structures and that produce catenulate spores with 
intercalary cells. This contrasts with the genus Aecidium Pers., which has a cup- 
shaped sorus with a well-developed peridium. Similar sori are found in the 
aecial state of Melampsora Castagne and the uredinia of Chrysomyxa Unger, 
Coleosporium Lév.,and other genera (Cummins & Hiratsuka 2003). The aecia of 
Phragmidium are (usually) Caeoma-type with catenulate spores or (less often) 
Uredo-type (Petrova & Denchev 2004) with verrucose or echinulate aeciospores. 
Cummins & Hiratsuka (2003) refer to the anamorphic genus Lecythea Léveillé 
the Phragmidium species with aecia corresponding to Caeoma UI of Sato & 
Sato (1985). Because Hennen et al. (2005) regard Lecythea as confusing and 
not in use, we consider Caeoma the appropriate anamorph for accommodating 
species with Phragmidium aecia. 

So far, 22 genera and 417 species of rust fungi have been described or reported 
from Pakistan (Afshan et al. 2008a,b,c,d, 2009a,b, 2010a,b, 2011a,b; Sultan et al. 
2008; Afshan & Khalid 2009; Khalid & Afshan 2009; Iqbal et al. 2008, 2009), 
including four genera of anamorphic rust fungi, Aecidium, Monosporidium 
Barclay, Peridermium (Link) J.C. Schmidt & Kunze, and Uredo Pers. Although 
no representatives of Caeoma have been reported previously from Pakistan, 
three new Caeoma species occurring on Rosa are described here. 


240 ... Afshan, Khalid & Niazi 


Materials & methods 

Freehand sections of infected tissue and spores were mounted in lactophenol and 
gently heated to boiling point. The preparations were observed under a NIKON YS 
100 microscope and photographed with a digipro-Labomed and a JSM5910 scanning 
electron microscope. Drawings of spores and paraphyses were made using a Camera 
Lucida (Ernst Leitz Wetzlar, Germany). An ocular micrometer was used for spore 
dimensions, with at least 25 spores measured for each spore stage. The rusted specimens 
have been deposited in the Botany Department herbarium, University of the Punjab, 
Lahore (LAH). 


Taxonomy 


Figs. A-C: Caeoma ahmadii (holotype), SEM micrographs. A: Aecidium containing 
aeciospores and paraphyses. B: Aeciospores. C: Detail of an aeciospore showing verrucose 
wall ornamentation (paraphyses visible in background). 


Caeoma spp, nov. (Pakistan) ... 241 


Caeoma ahmadii Afshan, Khalid & Niazi, sp. nov. Fies. A-E 


MycoBank MB 564295 
Differs from Phragmidium tuberculatum in larger aeciospores that are hyaline with 


orange-yellow contents. 
TyPE: PAKISTAN, KHYBER-PAKHTUNKHAWAH (KPK), Ayubia National Park, at 2135 m 


a.s.l., on Rosa microphylla Desf., stage I, 18 June, 2008, N.S. Afshan & A.N. Khalid. NSA 

#151A (Holotype, LAH NSA1114). 

ErymMoLoecy: Named after the world renowned mycologist, Sultan Ahmad. 
SPERMOGONIA, UREDINIA and TELIA not found. Agecta hypophyllous, on leaves, 


bright yellow to orangish yellow, rounded or oblong, scattered, 0.1-0.3 x 0.09- 
0.10 mm. PARAPHYSES numerous, erect to suberect, clavate, hyaline with light 


yellow granules, 50-70 um long and 8-12 um wide. AEcIOosPoREs globose to 


subglobose or ellipsoid to broadly ellipsoid, (23—)26-31 x 28-36 um; wall 1-1.5 
um thick, hyaline with orange-yellow contents, densely verrucose, with 5-8 


scattered pores. 


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ae: 
i. 


monemesemngahontene mt 
Sanne 
SSIS 


Figs. D-E: Caeoma ahmadii (holotype), lucida drawings. 
D: Aeciospores showing wall ornamentation. Scale bar= 9 um. 
E: Apices of paraphyses. Scale bar = 12 um. 


ComMENTs: Aeciospores of C. ahmadii are larger than those of Phragmidium 
tuberculatum J.B. Mull. (18-24 x 20-30 um), Ph. mucronatum (Pers.) Schtdl. 
(17-21 x 20-28 um), Ph. montivagum Arthur (16-19 x 21-26 um), Ph. rosae- 
arkansanae Dietel (16-23 x 23-27 um), Ph. speciosum (Fr.) Burrill (16-24 x 


242 ... Afshan, Khalid & Niazi 


24-35 um), and Ph. rosae-pimpinellifoliae Dietel (15-20 x 18-27 um). They also 
differ in color and wall ornamentation from those of Ph. tuberculatum. 


Fics. F-G: Caeoma khanspurense (holotype), lucida drawings. 
F: Apices of clavate paraphyses. Scale bar = 20 um. G: Mature aeciospores. Scale bar = 10 um. 


Caeoma khanspurense Khalid, Afshan & Niazi, sp. nov. Fics. F-G 
MycoBank MB 564317 


Differs from Phragmidium mucronatum and Ph. tuberculatum in larger aeciospores with 
verrucose wall ornamentation. 


Type: PAKISTAN, KHYBER-PAKHTUNKHAWAH (KPK), Helipad, Khanspur, at 2135 m 
a.s.l., on Rosa webbiana Wall. ex Royle, stage I, 23 May 2006, N.S. Afshan & A.N. Khalid. 
NSA #03 (Holotype, LAH NSA1116). 


ErymMo.ocy: Named after the type locality, Khanspur. 


SPERMOGONIA, UREDINIA and TELIA not found. Agecta hypophyllous or 
caulicolous, on the veins, stems and petioles, scattered, rounded or oblong, 
0.3-0.9 x 0.09-0.10 mm, orange yellow when fresh, surrounded by clavate 
paraphyses. AEcrosporEs globose, subglobose or ellipsoid to obovoid, pale 
yellow to orange-yellow, verrucose, occasionally with a plateau-shaped base, 
23-32 x 23-41 um, borne on one or two basal cells. PARAPHYSES clavate, 
hyaline, 70-105 um long, 8-12 um wide, with uniformly thin walls. 


ComMENTs: Aeciospores of C. khanspurense are larger than those of 
Phragmidium mucronatum (17-21 x 20-28 um) and Ph. tuberculatum (18-24 


Caeoma spp, nov. (Pakistan) ... 243 


x 20-30 um); their verrucose ornamentation further distinguishes them from 
the aeciospores of Ph. mucronatum. 

The broader aeciospores of C. khanspurense differ from those of C. rosicola 
(reported on the same host), characterized by verrucose to echinulate, 19-28 
um broad aeciospores. 

Aeciospores with a verrucose wall ornamentation with a plateau-shaped 
base distinguish C. khanspurense from Phragmidium rosae-pimpinellifoliae with 
smaller aeciospores (15-20 x 18-27 um). 


Fic. H: Caeoma rosicola, lucida drawing. 
Aeciospores. Scale bar = 10 um. 


Caeoma rosicola Afshan, Niazi & Khalid, sp. nov. Fics. H-J 
MycoBank MB 564318 
Differs from other Caeoma species in larger aeciospores and lack of paraphyses. 


Type: PAKISTAN, KHYBER-PAKHTUNKHAWAH (KPK), Helipad, Khanspur, at 2135 m 
a.s.l., on Rosa webbiana Wall. ex Royle, stage I, 23 May 2006, N.S. Afshan & A.N. Khalid. 
NSA #02 (Holotype, LAH NSA1117). 


ErymMo.Locy: Named after the host genus, Rosa. 


SPERMOGONIA, UREDINIA and TELIA not found. AEcta hypophyllous or 
petiolicolous, mostly on the veins, petioles, branches and fruits, causing 
malformations, yellowish orange to bright orange, scattered. PARAPHYSES 
not observed. AECIOSPORES globose to subglobose or ellipsoid to ovoid; wall 
hyaline with orange yellow contents, echinulate to verrucose, 19-28 x 21-41 
um, germ pores 1-4, mostly equatorial. 

ComMENTs: Larger aeciospores and the lack of paraphyses differentiate C. 


rosicola from other rust species reported on Rosaceae: Phragmidium rosae- 
pimpinellifoliae (15-20 x 18-27 um), Ph. rosae-arkansanae (16-23 x 23-27 um), 


244 ... Afshan, Khalid & Niazi 


ae 


Figs. I-J: Caeoma rosicola (holotype), SEM micrographs. I: Aeciospores. J: Detail of 
aeciospore showing echinulate wall ornamentation. 


Caeoma spp, nov. (Pakistan) ... 245 


Ph. mucronatum (17-21 x 20-28 um), and Ph. montivagum (16-19 x 21-26 
um). Phragmidium montivagum is further distinguished by aeciospores with 8 
scattered germ pores. 


Acknowledgements 

We are highly obliged to Higher Education Commission (HEC) of Pakistan for 
providing financial support. We sincerely thank Dr. Marcin Piatek (W. Szafer Institute of 
Botany, Polish Academy of Sciences, Poland) and Dr. Omar Paino Perdomo (Dominican 
Society of Mycology Santo Domingo, Dominican Republic) for their valuable suggestions 
to improve the manuscript and acting as presubmission reviewers. 


Literature cited 

Afshan NS, Khalid AN. 2009. New records of Puccinia and Pucciniastrum from Pakistan. Mycotaxon 
108: 137-146. http://dx.doi.org/10.5248/108.137 

Afshan NS, Berndt R, Khalid AN, Niazi AR. 2008a. New graminicolous rust fungi from Pakistan. 
Mycotaxon 104: 123-130. 

Afshan NS, Khalid AN, Javed H. 2008b. Further additions to the rust flora of Pakistan. Pakistan 
Journal of Botany 40(3): 1285-1289. 

Afshan NS, Khalid AN, Niazi AR. 2008c. New records of graminicolous rust fungi from Pakistan. 
Pakistan Journal of Botany 40(3): 1279-1283. 

Afshan NS, Khalid AN, Niazi AR. 2008d. New records and distribution of rust fungi from Pakistan. 
Mycotaxon 105: 257-267. 

Afshan NS, Iqbal SH, Khalid AN, Niazi AR. 2009a. A new anamorphic rust fungus with a new 
record of Uredinales from Azad Kashmir, Pakistan. Mycotaxon 112: 451-456. 
http://dx.doi.org/10.5248/112.451 

Afshan NS, Khalid AN, Iqbal SH, Niazi AR, Sultan A. 2009b. Puccinia subepidermalis sp. nov. and 
new records of rust fungi from Pakistan. Mycotaxon 110: 173-182. 
http://dx.doi.org/10.5248/110.173 

Afshan NS, Khalid AN, Niazi AR. 2010a. Three new species of rust fungi from Pakistan. Mycological 
Progress http://dx.doi.org/10.1007/s11557-010-0655-8 

Afshan NS, Khalid AN, Iqbal SH, Niazi AR, Sultan A. 2010b. Puccinia anaphalidis-virgatae, a new 
species and a new variety of rust fungi from Fairy Meadows, Northern Pakistan, Mycotaxon 
112: 483-490. http://dx.doi.org/10.5248/112.483 

Afshan NS, Khalid AN, Iqbal SH, Niazi AR. 201 1a. Puccinia species new to Azad Jammu & Kashmir, 
Pakistan, Mycotaxon: 116: 175-182. 

Afshan NS, Khalid AN, Niazi AR, Iqbal SH. 2011b. New records of Uredinales from Fairy Meadows, 
Pakistan. Mycotaxon: 115: 203-213. http://dx.doi.org/10.5248/115.203 

Cummins GB, Hiratsuka Y. 2003. Illustrated genera of rust fungi. Third ed. The American 
Phytopathological Society. APS Press, St. Paul, MN. 

Hennen JF, Figueir edo MB, Carvalho AA, Hennen PG. 2005. Catalogue of the species of plant rust 
fungi (Uredinales) of Brazil. FAPESP, CNPq, FAPERJ, NSF, USDA. 

Iqbal SH, Khalid AN, Afshan NS, Niazi AR. 2008. Rust Fungi on Saccharum species from Pakistan. 
Mycotaxon 106: 219-226. 

Iqbal SH, Afshan NS, Khalid AN, Niazi AR, Sultan A. 2009. Additions to the rust fungi of Fairy 
Meadows, Northern Areas of Pakistan. Mycotaxon 109: 1-7. http://dx.doi.org/10.5248/109.1 


246 ... Afshan, Khalid & Niazi 


Khalid AN, Afshan NS. 2009. Additions to the graminicolous rust fungi of Pakistan. Mycotaxon 
108: 175-183. http://dx.doi.org/10.5248/108.175 

Petrova RD, Denchev CM. 2004. A taxonomic study of Phragmidiaceae (Uredinales) in Bulgaria. 
Mycologia Balcanica 1: 95-115. 

Sato T, Sato S. 1985. Morphology of the aecia of the rust fungi. Trans. Br. Mycol. Soc. 85: 223-238. 

Sultan MA, Haq I, Khalid AN, Mukhtar H. 2008. Two new anamorphic rust fungi from northern 
areas of Pakistan. Mycotaxon 105: 23-27. 


ISSN (print) 0093-4666 © 2012. Mycotaxon, Ltd. ISSN (online) 2154-8889 


MYCOTAXON 


http://dx.doi.org/10.5248/120.247 
Volume 120, pp. 247-251 April-June 2012 


Nomenclatural status and morphological notes on 
Tubifera applanata sp. nov. (Myxomycetes) 


D.V. LEONTYEV’™ & K.A. FEFELOV’ 


‘Department of Biotechnology, Kharkiv Zooveterinary Academy, 
Akademichna str. 1 Mala Danylivka, Kharkiv 62341 Ukraine 
*The Institute of Plant and Animal Ecology, 
Vosmogo Marta str. 202, Yekaterinburg 620144 Russia 
* CORRESPONDENCE TO: protista@mail.ru 


Asstract — Tubifera applanata sp. nov. is proposed to validate “Tubulifera applanata” nom. 
inval. The species diagnosis and some notes on its morphology are provided. At 0.40-0.65 mm 
diam., individual sporothecae are somewhat larger than in T. ferruginosa and T. microsperma 
and smaller than those in T. casparyi. Circular ornamentations on the inner peridial surface 
in T: applanata are larger than previously noted, reaching a size up to 2.9 um. 


Key worps — morphometry, SEM, species delimitation 


Introduction 

In 2001, C. Lado stated that the invalid generic name “Tubulifera” O.F. Mull. 
1775 had been validated as Tubulifera O.F. Mill. ex Jacq. 1779, a name with 
priority over Tubifera J.F. Gmel. 1792 (Lado 2001). On this understanding, 
Leontyev & Fefelov (2009) proposed a new species, Tubulifera applanata. 

Subsequently, Lado (2011) indicated that, contrary to previous opinion, 
Jacquin’s publication failed to validate the genus “Tubulifera.” This means that 
all species names published in “Tubulifera,” including “Tubulifera applanata,” 
are also invalid (ICBN [Vienna Code] Article 43.1). We therefore propose a 
validation of “Tubulifera applanata” as Tubifera applanata sp. nov. and provide 
additional morphological data. 


Materials & methods 

A total of 13 specimens of Tubifera applanata were examined (CWU MR 001, 038a, 
038b, 039, 054, 058, 074, 077, 121, 122, 123, 125, 126). Additionally, 8 specimens of 
T. ferruginosa (Batsch) J.E. Gmel. (CWU MR 006, 023, 012, 091, 095, 103, 113, 114), 4 of 
T. microsperma (Berk. & M.A. Curtis) G.W. Martin (CWU MR 008, 041, 049, 153), and 
3 of T: casparyi (Rostaf.) T. Macbr. (CWU MR 049, 181, 183) were used for comparison. 
All the specimens were collected during 2003-2010 in the East Forest-Steppe region 


248 ... Leontyev & Fefelov 


of Ukraine (Gomolsha Forests National Nature Park, Kharkiv region). Material was 
deposited in the herbarium of the V.N. Karasin National University of Kharkiv (CWU), 
the subsection of Reticulariaceae (MR). 

The specimens were examined using a stereoscopic microscope MBS-9 and scanning 
electron microscope (SEM) Jeol JSM-6060 (30 kV, gold evaporation). 

The diameters of intact closed sporothecae were measured from above by a 
micrometer. After drawing an imaginary line corresponding to the largest fruit body 
diameter on the surface of the pseudoaethalium, we measured 30 sporothecae along this 
line from the edge of the pseudoaethalium. The diameters of annular ornamentations on 
the inner peridial surface were measured using the SEM scale. 

Means, standard deviations, and standard errors were calculated using Stat Soft 
Statistica 8.0 and Microsoft Excel 2003 software to describe the morphological variety. 


Results & discussion 


Tubifera applanata Leontyev & Fefelov, sp. nov. FIG. 1c-D 
MycoBank MB 561713 
“Tubulifera applanata” Leontyev & Fefelov, Bol. Soc. Mycol. Madrid 33: 119. 2009 


Differs from Tubifera ferruginosa by its large pseudoaethalia, flat sporothecal tips, 
annular embossments on the internal peridial surface, and salmon color of young 
fructifications. 


Type: Ukraine, Kharkiv region, Gomolsha Forests National Nature Park, Zadinetske 
forestry, edge of pine forest near ‘Bile Ozero’ Camp (49°36'50.69"N 36°21'06.15”E), on 
the bark of fallen trunk of Pinus sylvestris L., 14.07.2003, leg. D.V. Leontyev (Holotype, 
CWU MR 039). 


EryMmo oey: applanatus (Lat.) = flat; referring to the flattened sporothecal tips. 


PSEUDOAETHALIUM large, (12—)23-37(-73) mm long, (7-)16.5-27.5(-38) mm 
wide, 4-6 mm in height, oval or irregular as seen from above, flat pulvinate, rust- 
brown. SPOROTHECAE Straight, clearly prismatic from mutual pressure. Tips 
OF SPOROTHECAE 0.4—0.65 mm diam., flat or slightly convex, 5-6 angular or 
almost circular, equal in height, forming a smooth surface of pseudoaethalium. 
PERIDIUM opaque, light brown, lateral walls of the sporothecae often plicate. 
INTERNAL SURFACE OF PERIDIUM covered with annular embossments 
(0.4-)1.5-2.9 um diam., quite distant from each other. COLUMELLA absent 
or not reaching the top of the sporothecae, thick, with color of spore mass. 
PSEUDOCAPILLITIUM absent or scanty, filiform, appearing as occasionally 
branched strings. HyPOTHALLUS spongy, poorly developed or absent. SPORES 
in mass rust-brown, brownish by transmitted light, 4.9-6.3(-7.5) um diam., 
banded reticulate. PLAsmopIUM salmon or rusty-cream. In coniferous forests, 
on bark, litter and slightly decomposed wood. 


Notes on the morphology 

Tubifera applanata is characterized by the prismatic shape of the individual 
sporothecae, their flat hexagonal tips, ring-like ornamentations on the inner 
peridial surface, and the salmon color of young fructifications. Although we 


Tubifera applanata sp. nov. ... 249 


first noted that quantitative parameters such as pseudoaethalium size and 
spore diameter distinguished T: applanata from related species (Leontyev & 
Fefelov 2009), we did not realize that individual sporothecal diameters might 
also serve as a diagnostic criterion. Moreover, there were no data referring to 
the diameter of sporothecae in other Tubifera species (Nannenga-Bremekamp 
1991, Neubert et al. 1993, Ing 1999). 

The study of sporothecae size in Tubifera applanata, T: ferruginosa, T. micro- 
sperma, and T. casparyi has shown that all species have significant differences 
(p<0.01) from one another. Measurements show that individual sporothecae 
are somewhat larger in T: applanata than in T. ferruginosa and T. microsperma 
and smaller than those in T’ casparyi (TABLE 1). 


TABLE 1. Sporothecal diameters in four Tubifera species. 


T. applanata T. ferruginosa T. microsperma T. casparyi 


Range of specimen means (1m) | 464-633. =| = 322-474 330-398 512-692 


Mean (um) 


Standard deviation [sd] (tm) 


Rounded variation, for use in species 
descriptions (mm) 


04-065 | 03-05 | 03-045 | 0.5-0.7 


Although the difference in sporothecae size between T. applanata and 
T. ferruginosa is quite small (0.1-0.2 mm), it can be seen with the naked eye. 
We propose to use this criterion as an additional character in descriptions of 
Tubifera species. 


Peridium ultrastructure 

Nelson et al. (1982) introduced a new diagnostic criterion into Tubifera 
taxonomy — the type of inner peridial ornamentation, noting that in T’ micro- 
sperma the inner surface of the peridium is ornamented by so-called ‘rimmed 
craters. In Ukrainian specimens of T: microsperma, the ornamentation agrees 
with this description (Fic. 1A-B). 

In 2009 the SEM-study of T: applanata showed ‘peridial rings, similar to 
those of T! microsperma but much lower and more distant from each other 
(Leontyev & Fefelov 2009). Both ‘peridial rings’ and ‘rimmed craters’ appear to 
be remnants of channels used by the plasmodium for active water evacuation 
(R. McHugh, pers. comm.). 

We previously indicated the outer ‘peridial ring’ diameter in T’ applanata as 
0.4-0.9 um (Leontyev & Fefelov 2009). Improved SEM techniques demonstrate 
that in the type specimen, these structures reach 1.6-2.9 um diam. (FIG. 1C-D), 
significantly larger than similar structures in T. microsperma. In T: applanata, 
rings < 1 um diam. are usually less prominent than the larger ones. 


250 ... Leontyev & Fefelov 


ZE9R8 Wit 


hI 


Figure 1. A-B: Tubifera microsperma [CWU MR 135]—ornamentation of the inner peridium 
surface illustrating ‘rimmed craters’ C-D: Tubifera applanata [CWU MR 039, holotype]— 
ornamentation of the inner peridium surface illustrating ‘peridial rings’ and intricate filaments. 


Tubifera applanata sp. nov. ... 251 


This study has shown that the peridial surface of T. applanata is ornamented 
by thin intricate filaments, appearing to be composed of dried slime (Fic. 1D). 
Such filaments are especially abundant in the bottom of the ‘peridial rings’ 


Acknowledgments 

We express our sincere gratitude to David W. Mitchell (East Sussex, England), 
Carlos Lado (Royal Botanic Garden, Madrid, Spain), and Shaun Pennycook (Landcare 
Research, Auckland, New Zealand) for valuable comments and corrections; and to 
Yana V. Belousova (Kharkiv State Zooveterinary Academy) for the assistance in the 
measurements of sporothecae. 


Literature cited 

Ing B. 1999. The Myxomycetes of Britain and Ireland. An identification handbook. Richmond. 

Lado C. 2001. Nomenmyx: a nomenclatural taxabase of Myxomycetes. Cuad. Trab. Fl. Micol. Iber. 
1621-221. 

Lado C. 2011. The nomenclatural status of the genus Tubifera (Myxomycetes). Taxon 60: 221-222. 

Leontyev DV, Fefelov KA. 2009. Tubulifera applanata. The new species from Eastern Europe and 
Northern Asia. Bol. Soc. Mycol. Madrid 33: 115-127. 

Nannenga-Bremekamp NE. 1991. A guide to temperate Myxomycetes. Biopress Ltd., Bristol. 

Nelson RK, Scheetz RW, Alexopoulos CJ. 1982. Taxonomic studies in the myxomycetes. V. 
Significance of peridial and spore ornamentations in the genus Tubifera, with a revised key to 
the species. Mycologia 74: 541-548. http://dx.doi.org/10.2307/3792740 

Neubert H, Nowotny W, Baumann K. 1993. Die Myxomyceten Deutschlands und des angrenzenden 
Alpenraumes unter besonderer Beriicksichtigung Osterreichs. Bd. 1. Gomaringen, Baumann 
Verlag. 


ISSN (print) 0093-4666 © 2012. Mycotaxon, Ltd. ISSN (online) 2154-8889 


MY COTAXON 


http://dx.doi.org/10.5248/120.253 
Volume 120, pp. 253-267 April-June 2012 


Paralepistopsis gen. nov. and Paralepista 
(Basidiomycota, Agaricales) 


ALFREDO VIZZINI* & ENRICO ERCOLE 


Dipartimento di Scienze della Vita e Biologia dei Sistemi - Universita degli Studi di Torino, 
Viale Mattioli 25, I-10125, Torino, Italy 
*CORRESPONDENCE TO: alfredo. vizzini@unito. it 


ABSTRACT — Paralepistopsis, a new genus in Agaricales, is proposed for the rare toxic species, 
Clitocybe amoenolens from North Africa (Morocco) and southern and southwestern Europe 
and C. acromelalga from Asia (Japan and South Korea). Paralepistopsis is distinguished from 
its allied clitocyboid genera by a Lepista flaccida-like habit, a pileipellis with diverticulate 
hyphae, small non-lacrymoid basidiospores with a smooth slightly cyanophilous and 
inamyloid wall, and the presence of toxic acromelic acids. Combined ITS-LSU sequence 
analyses place Paralepistopsis close to Cleistocybe and Catathelasma within the tricholomatoid 
clade. Our phylogenetic analysis further supports Lepista subg. Paralepista (= Lepista sect. 
Gilva) as an independent clitocyboid evolutionary line. We recognize the genus Paralepista, 
for which we propose twelve new combinations. 


KEY worps — Agaricomycetes, erythromelalgia/acromelalgic syndrome, Clitocybe sect. 
Gilvaoideae, /catathelasma clade 


Introduction 

The genus Clitocybe (Fr.) Staude traditionally encompassed saprobic agarics 
that produce fleshy basidiomata with often adnate-decurrent lamellae, convex 
to funnel-shaped pilei, usually a whitish to pinkish yellow spore print, and 
smooth non-amyloid basidiospores (Kihner 1980, Singer 1986, Bas 1990, 
Raithelhuber 1995, 2004). 

Recent molecular studies that included a significant number of Clitocybe 
species (Moncalvo et. al. 2002, Redhead et al. 2002, Matheny et al. 2006, Vizzini 
et al. 2010a,b, 2011) have shown that taxa in this traditional genus do not form 
a monophyletic group but rather a heterogeneous artificial set of disparate and 
(in many cases) phylogenetically unrelated taxa (the so-called clitocyboid fungi 
or Clitocybe s.l.). 

Clitocybe amoenolens is a rare and rather localized species known thus far 
only from Morocco, southern France, northern and central Spain, and central 


254 ... Vizzini & Ercole 


Italy (Malencon & Bertault 1975, Bon 1987, Poumarat & Neville 1993, Contu 
et al. 1999, Moreau et al. 2001, Martinez et al. 2010). It was responsible, first 
in France (Fourré 1997, Charignon & Garcin 1998, Moreau et al. 2001, Saviuc 
et al. 2001, 2002) and then in Italy (Leonardi et al. 2002, Marinetti & Recchia 
2005), for induced erythromelalgia (= acromelalgia syndrome sensu Saviuc et 
al. 2001), a poisoning syndrome caused by the ingestion of C. acromelalga in 
Japan (Nakamura et al. 1987). This syndrome is characterized by varying degrees 
of tingling sensations, followed by intense burning pain in the extremities 
but predominantly in the feet (Saviuc & Danel 2006). Clitocybe amoenolens 
was confused with edible mushrooms in the Lepista flaccida complex (e.g., 
L. flaccida (Sowerby) Pat., L. lentiginosa (Fr.) Bresinsky, L. gilva (Pers.) Pat.) and 
with Infundibulicybe gibba (Pers.) Harmaja (Fourré 1997, Moreau et al. 2001). 

Clitocybe amoenolens shows features intermediate between Clitocybe s.s. 
(smooth spores) and Lepista subg. Paralepista (cream spore-print, spotted 
pileus, lamellae separable from context, and cyanophilic spores released in 
tetrads), making its generic position uncertain. 

Using recent French and Italian collections of C. amoenolens, we investigated 
its phylogenetic position within the clitocyboid fungi through morphological 
and molecular analyses and expanded its known geographic distribution. 


Materials & methods 


Morphology 

Macromorphological features were described from fresh specimens. Microscopical 
preparations from dried material were rehydrated in 3% KOH and stained in Congo red, 
Cresyl Blue, Cotton Blue and Melzer’s reagent. 

Basidiospore measurements are based on means of 120 spores from prints (four 
collections), stained in Melzer’s reagent. The basidia width was measured at the 
widest part and the length from the apex (sterigmata excluded) to basal septum. The 
following abbreviations are used in text: L = number of entire lamellae; | = number of 
lamellulae between each pair of entire lamellae; Q = the quotient of length and width 
of the spores in side view; Qm = average quotient. Colour terms in capital letters (e.g. 
Pale Ochraceous-Buff) are those of Ridgway (1912). Herbarium acronyms follow 
Thiers (2011). Author citations follow Index Fungorum (http://www.indexfungorum. 
org/authorsoffungalnames.htm). All examined collections are housed at TO. The new 
genus and new combinations are deposited in MycoBank (http://www.mycobank.org/ 
DefaultPage.aspx ). 


DNA extraction, PCR amplification, and DNA sequencing 

Genomic DNA was isolated from 1 mg of herbarium material (TaBLE 1) using the 
DNeasy Plant Mini Kit (Qiagen, Milan Italy) according to the manufacturer’s instructions. 
Universal primers ITS1F/ITS4 were used for the ITS region amplification (White et al. 
1990, Gardes & Bruns 1993) and primers LROR/LR7 for the LSU rDNA amplification 
(Vilgalys & Hester 1990, Vilgalys lab unpubl., http://www.botany.duke.edu/fungi/ 


Paralepistopsis gen, nov. and Paralepista ... 255 


TABLE 1. Collections newly sequenced in this study. 


GENBANK ACC. NUMBERS SOURCE, COUNTRY. 
> > 


pREClE® ITS LSU DATE, COLLECTOR 
Paralepistopsis amoenolens 1 JQ585653 JQ585654 if Reuerics Pe st 
Paralepistopsis amoenolens 2 JQ585655 — 2 Pena ree a 
Paralepista flaccida 1 JQ585656 JQ585657 a Ron te ake 
Paralepista flaccida 2 JQ585658 JQ585659 ae en 
Paralepista gilva JQ585660 JQ585661 eee eaten 


09/11/2011, A. Vizzini 


mycolab). Amplification reactions were performed in a PE9700 thermal cycler (Perkin- 
Elmer, Applied Biosystems) following Vizzini et al. (2010b). PCR products were purified 
with the AMPure XP kit (Beckman) and sequenced by DINAMYCODE srl (Turin, Italy) 
and MACROGEN Inc. (Seoul, Republic of Korea). Sequences were assembled and edited 
with the phred/phrap/consed software suite. The sequences were submitted to GenBank 
and their accession numbers are reported in TABLE | and Ficure 1. 


Sequence alignment and phylogenetic analysis 

The sequences obtained in this study were checked and assembled using Geneious 
v5.3 (Drummond et al. 2010) and compared to those available in GenBank (http://www. 
ncbi.nlm.nih.gov/Genbank/) using the blastn algorithm. Based on the blastn results, 
sequences were selected according to the outcomes of recent phylogenetic studies on 
Agaricales (Matheny et al. 2006, Binder et al. 2010, Vizzini et al. 2011). A combined 
ITS and LSU sequence analysis was carried out using sequences from the same strain 
or specimen. Xeromphalina campanella (GU320006 and GU320009) was used as 
outgroup. Alignments were generated using MAFFT (Katoh et al. 2002) with default 
conditions for gap openings and gap extension penalties. The sequence alignment, its 
manual adjustment, and the best-fit models estimation follow Vizzini et al. (2010b). 
The GTR+G and GTR+G substitution models were used in the ITS and LSU analyses, 
respectively. A partitioned matrix was used in all the analyses. Molecular-phylogenetic 
analyses were performed using the Bayesian Inference (BI) and Maximum Likelihood 
(ML) approaches. BI using Monte Carlo Markov Chains (MCMC) was carried out 
with MrBayes 3.1.2 (Huelsenbeck and Ronquist 2001). Four incrementally heated 
simultaneous MCMC were run over 5.000.000 generations, under model assumption. 
Trees were sampled every 500 generations resulting in an overall sampling of 10.001 
trees. The “burn-in” value was evaluated using Tracer 1.5 (Rambaut & Drummond 2007). 
The first 20% of trees was discarded as “burn-in”. For the remaining trees, a majority rule 
consensus tree showing all compatible partitions was computed to obtain estimates for 
Bayesian Posterior Probabilities (BPP). ML estimation was performed through RAxML 
v.7.0.4 (Stamatakis 2006) with 1.000 bootstrap replicates (Felsenstein 1985) using the 


256 ... Vizzini & Ercole 


LL ema Callistosporium graminicolor 0Q484065, AY745702 
Callistosporium xanthophyllum AF325667, AF261406 

— Callistosporium luteoolivaceum AF 325666, AF261405 

0.9/- Pseudoomphalina pachyphylla Hm191750, # 


oe Entoloma abortivum F 3940730, AF223169 


4/100 Entoloma sinuatum Gu289652, AY207199 
Less —— = —__ Entoloma cetratum £U784213, AY700180 

Entoloma prunuloides 0206983, AY691891 
199 citopilus giovanellae £F 413030, EF413027 

Clitopilus prunulus 0202272, AY700181 
4/100 ->——- Rhodocybe fallax AF357018, AF223165 
Rhodocybe mundula Da494694, AY700182 
400 ->—— Hypsizygus tessulatus Da917653, DQ917664 
—— Ossicaulis lignatilis 09825426, AF261397 
Tricholomella constricta 0Q825429, AF223188 
Lyophyllum palustris AF357043, AF223199 
a7 Lyophyllum sykosporum AF357051, AF357073 
i Lyophyllum boudieri 00825427, 00825430 
atoo Clitocybe rhizophora JF907812, # 

Clitocybe vermicularis JF907813, # 
— Clitocybe vermicularis JF907817, # 


400 


4/100 


4/100 


Clitocybe subditopoda 0a202269, AY691889 

litocybe candicans DQ202268, AY645055 

1100 r Clitocybe puberula FM877682, FM877681 
Clitocybe puberula FM877683, FM877680 
Clitocybe ditopa JF907805, # 
Clitocybe phaeophthalma Fms77684, FM877679 
-/95¢ Clitocybe odora #, AY206714 
0.92/- Clitocybe phyllophila #, AY207157 
Clitocybe ruderalis #, AY207159 
Collybia tuberosa AF274376, AF261385 
0.84). Clitocybe fragrans JF907811, AY207153 
er Lepista nuda AF357062, AY207223 
a Lepista personata AF241522, # 7 7 

epista saeva #, AY207224 

rel Lepista Sordida FJ770391, AY207225 Clitocybe/L epista 
{C Clitoc ‘be metachroa JF907806, AY207155 

ar Clitocybe vibecina JF907821, AY207160 
09507 -— Lepista irina 0Q221109, DQ234538 


0.76/- (= Se ees Lepista Ovispora #, GQ289207 


Clitocybe dealbata AF357061, AF223175 
70 Clitocybe nebularis AF357063, AF223217 
L_usoil Gitocybe nebularis DQ486691, DQ457658 
Clitocybe nebularis AY521248, AY586685 
1n00+ Clitocybe amoenolens 1 JQ585653, JQ585654 


sr i Paralepistopsis 
223§ "coy teu amoenotens 2 sastets pistopsis | /catathelasma clade 


Cleistocybe vernalis EF416917, EF416916 
re 


Catathelasma imperiale Gag81498, AF261402 
Catathelasma ventricosum 0a486686, DQ089012 
Tricholoma focale AF319437, TFU76460 
Tricholoma imbricatum AF319426, TIU76458 
Lise Tricholoma pardinum AF319427, TPU76462 

Tricholoma venenatum AF319433, TVU76463 
Neohygrophorus angelesianus 00494678, DQ470814 
—f__,_,, Clitocybe alkaliviolascens JF907807, # 
100, Infundibulicybe gibba Da490635, DQ457682 
Clitocybe geotropa #, AY207154 
4 Omphalina pyxidata JN944402, JN944403 
Pseudoclitocybe cyathiformis JF926522, JF926523 


0.6107 
L___ioof '— Pseudoclitocybe expallens JF926524, JF926525 
ove M 
197 


1/99 


193 


lusumecia bettlakensis JF926520, JF926521 


4/98 pepista faccida #, eNeelerd 
epista flaccida 2 40585658, JQ585659 H 
-— Lepista Haccids 1 JOBSSESH: JQ585657 | P. are al lepista 
7 1/100" ——————- Lepista gilva JQ585660, JQ585661 
4/400 Cc  Arthenia auriscalpium AAU66428 (ITS and LSU fragments) 
0.99/61 ; Arrhenia lobata ALU66429 (ITS and LSU fragments) 
400 -——— My cena galericulata Da404392, AY647216 


Mycena plumbea 00494677, DQ470813 


Xeromphalina campanella Gu320006, GU320009 


0.04 expected change per sites 


FicurE 1. Tricholomatoid clade. Bayesian phylogram obtained from the combined ITS-LSU 
sequence alignment. Support values for clades that are supported in either the Bayesian (Posterior 
Probabilities values - BPP) or Maximum likelihood (ML Bootstrap percentage - MLB) analyses 
are indicated. BPP > 0.70 and MLB > 50% are given above branches. Numbers (1, 2) refer to the 
Paralepistopsis and Paralepista collections reported in TABLE 1. 


GTRGAMMA algorithm for both ITS and LSU to perform a tree inference and search 
for a good topology. Support values from bootstrapping runs (MLB) were mapped on 
the globally best tree using the “-fa” option of RAxML and “-x 12345” as a random seed 
to invoke the novel rapid bootstrapping algorithm. Only BPP values over 0.70 and MLB 
over 50% are reported in the resulting tree (Fic. 1). Pairwise % identity values of ITS 
sequences were calculated using MEGA 5.0 (Tamura et al. 2011). 


Results 


Molecular results 

The combined dataset comprises a total of 71 taxa (including 66 from 
GenBank) and is 2412 base pairs long. The ITS and LSU datasets are 796 and 
1616 base pairs long, respectively. Topologies of the combined ITS and LSU 
Bayesian and Maximum Likelihood trees are congruent (Fic. 1). 

In both analyses the two Clitocybe amoenolens collections (ITS pairwise 
identity value = 99.9%) clearly cluster with the C. acromelalga collection 
(BPP 1 and MLB 100%) in the /catathelasma clade, a monophyletic group 


Paralepistopsis gen, nov. and Paralepista ... 257 


in the tricholomatoid clade (Matheny et al. 2006, Ammirati et al. 2007). The 
ITS pairwise identity value between the C. acromelalga and C. amoenolens 
sequences is 94.4%; accepting an intraspecific variability lower than 3% (Nilsson 
et al. 2008), C. amoenolens and C. acromelalga should be considered distinct 
species. Cleistocybe vernalis Ammirati et al. cluster sister to the C. acromelalga/ 
C. amoenolens pair (BPP 0.82); Catathelasma Lovejoy is basal to this group with 
low BPP and MLB values. 

Clitocybe amoenolens and C. acromelalga have no phylogenetic relationship 
with other Clitocybe species. Accordingly, we propose to establish for them a 
new genus, Paralepistopsis. 

Lepista flaccida and L. gilva are not phylogenetically related to the other 
Lepista species. 


Taxonomy 


Paralepistopsis Vizzini, gen. nov. 
MycoBank MB 564340 
A Paralepista differt sporis levis, haud subglobosis, atque praesentia acidi acromelalgici 


qua de causa venenatae species sunt. et in stuctura molecularis (ITS-spatiis internis 
transcriptis et LSU DNA). 


TYPE SPECIES — Clitocybe amoenolens Malencon 


EryMOLoGy — named in reference to its resemblance to Paralepista species. 


Basidiomata agaricoid (with distinct pileus, lamellae and stipe), resembling 
those of Paralepista gilva, veils absent, spore-print whitish to cream, 
basidiospores thin-walled, smooth, inamyloid and slightly cyanophilous, pileal 
surface a cutis of repent to interwoven, cylindrical hyphae, clamp-connections 
present, no sarcodimitic texture in any part of the basidioma. On the ground, 
never on wood. 


Paralepistopsis amoenolens (Malencon) Vizzini, comb. nov. Fic. 2 
MycoBank MB 564341 
= Clitocybe amoenolens Malencon, in Malencon & Bertault, Flore des champignons 
superieurs du Maroc 2 - Trav. Inst. Sci. Chérifien, Sér. Bot. Biol. Vég. 33: 141 (1975). 
SELECTED DESCRIPTIONS — Malencon & Bertault (1975: 138-141); Moreau et al. (2001: 
99-100, 101-103). 
SELECTED ICONOGRAPHY — Malencon & Bertault (1975: pl. 8); Poumarat & Neville 
(1993: 48); Martinez et al. (2010: fig. 2, p. 104). 
PitEus (2-)3.5-7(-8) cm diam., fleshy, sub-elastic, hemispherical to convex 
at first, becoming plano-convex and applanate, finally plano-concave, at times 
broadly umbonate; margin narrowly inrolled and decurved at first, remaining 
inrolled for a long time, then expanding to become somewhat wavy, shortly 
sulcate-striate or corrugated; not hygrophanous, occasionally appearing 


258 ... Vizzini & Ercole 


hygrophanous when water soaked; surface slightly viscid when moist, at first 
entirely whitely pruinose, then pubescent-pruinose only at margin, typically 
diffracted-scaly near the disc, sometimes corrugated, wrinkled and areolate, 
later subglabrous often with more or less concentrically arranged watery, drop- 
like spots, especially near margin (as many Lepista species); at first cream-beige 
coloured (Capucine Buff, Pale Ochraceous-Salmon, Pale Ochraceous-Buff), 
then pinkish beige to rusty orange (Ochraceous-Buff, Zinc Orange) towards 
the centre. LAMELLAE crowded to close (L = 40-48(-50), 1 = (0—)1-2(-3)), 
thick, interspersed with lamellulae, (2-)3-4(-5) mm broad, decurrent, easily 
separable from the pileus context, at times intervenose or forked towards the 
stipe, at first whitish then yellowish ochre (Ivory Yellow, Chamois) to pinkish 
beige (Pale Ochraceous-Salmon, Pale Ochraceous-Buff); edges even, entire, 
concolorous. STIPE (2.5—)3-3.5(-5) cm long, 0.7-1.3 cm thick, short, central 
or subexcentric, equal or with a somewhat enlarged base, straight or recurved, 
concolorous with the pileus or slightly paler, minutely white pruinose at apex, 
glabrous elsewhere, stuffed with white medulla, becoming hollow; the base 
often with copious whitish tomentum with adhering Pinaceae needles and 
woody debris. CONTEXT 4-11(-14) mm thick at disc, elastic, white in the 
pileus, whitish cream (Capucine Buff) in the stipe cortex, unchanging; taste 
mild, fungoid, subfarinaceous, slightly bitter-farinaceous after long mastication; 
odour strong, aromatic, floral, reminiscent of Inocybe corydalina, Tricholoma 
caligatum, Lepista irina, or Entoloma ameides. SPORE PRINT whitish to pale 
cream (Light Buff, Pale Pinkish Buff). 

BASIDIOSPORES (3.8—)4.0-5.4(-5.6) x (2.3-)3.2-4.0(-4.3) um (n = 120), 
on average 4.8 x 3.4 um, Q = 1.3-1.7, Qm = 1.42, broadly ellipsoid, hyaline, 
thin-walled, smooth, slightly cyanophilous, inamyloid, non-dextrinoid, usually 
with only a single oil drop and a distinct truncated apiculus up to 0.7 um 
long, mostly adhering together in tetrads in dried specimens. Basrip1A (25-) 
30-37(-38) x 5-6(-7) um, cylindro-clavate, usually four-spored, occasionally 
two-spored, sterigmata up to 5 um long. HYMENOPHORAL TRAMA regular in 
young stages, but subirregular in mature basidiomata, consisting of hyaline, 
elongated, cylindrical hyphae 4-6(-8) um broad. PLEUROcystTiD1A absent. 
LAMELLA EDGES fertile, with rare, scattered cells, not well differentiated from 
basidia, 15-45(-60) x 2.5-5(-7) um, cylindric to subfusiform or sublageniform, 
often curved and flexuous, sometimes forked at apex, hyaline, thin-walled. 
PILEIPELLIS duplex: upper layer (suprapellis) a soon disappearing, slightly 
gelatinous thin cutis (10-30 um thick), of cylindrical hyphae, 1-3 um broad; 
lower layer (subpellis) (150-350 um thick) composed of densely arranged 
parallel to slightly interwoven hyphae (4-6 um in diam.), terminal elements 
scattered, erect and repent cylindrical to fusiform-lageniform, sometimes 
with short lateral outgrowths. PILEITRAMA consisting of cylindrical or slightly 


Paralepistopsis gen, nov. and Paralepista ... 259 


\ 


FiGuRE 2. Paralepistopsis amoenolens. a. Basidiomata. b. Diffracted-scaly pileus with minute 
squamules. c. Areolate-corrugated pileus. d. Sulcate-striate pileus margin (red arrow), lamellae, 
and stipe. e. Basidiomata. [a—b from TO AV2007; c-d from TO AV2006 ; e from TO AV2004. 
Bars = 1 cm] 


260 ... Vizzini & Ercole 


inflated, smooth hyphae, (4-)5-9(-12) um broad, irregularly arranged, and 
with aeriferous lacunae. STIPITIPELLIS a cutis consisting of hyphae 3-4 um 
broad; towards the stipe apex with numerous thin-walled, flexuous, cylindrical 
to subfusiform caulocystidioid elements, 20-35 x 3-6 um. STIPITITRAMA 
formed by hyphae 6-7 um broad. THROMBOPLEROUS HYPHAE (= oleiferous 
hyphae sensu Clémencon 2004) present especially in subpellis, pileus and 
hymenophoral trama. PIGMENT (yellowish) usually parietal and intracellular 
(cytoplasmatic) in the pileipellis; sometimes minutely incrusting or forming 
extracellular masses and granules. CLAMP CONNECTIONS present at nearly all 
septa. 

HABITAT & DISTRIBUTION: scattered, gregarious, occasionally subcaespitose 
on pinaceous needle-beds and debris, often together with Lepista flaccida, on 
calcareous soil. Autumn. Very rare; known only from Morocco, France, Spain, 
and Italy. 

MATERIAL STUDIED: FRANCE, SAvorgE, HAUTE-MAURIENNE VALLEY, Lanslebourg- 

Mont-Cenis, 02 Sept 2011, litter of Picea abies, 1400 m asl, leg. G. Moretto (TO AV2004); 

14 Oct 2011, leg. G. Moretto (TO AV2005). - ITALY, PrEDMONT, HiGH SusaA VALLEY, 

Salbertrand, Parco Naturale del Gran Bosco di Salbertrand, 20 Oct 2011, litter of Picea 

abies, 1500 m asl, leg. A. Vizzini (TO AV2006); Sauze d’Oulx, Parco Naturale del Gran 

Bosco di Salbertrand, 12 Nov 2011, litter of Larix decidua, 1550 m asl, leg. S. Anselmino 

(TO AV2007). 
Clitocybe amoenolens was originally described from Morocco, in the Middle 
Atlas, growing among Cedrus atlantica litter in a high-altitude cedar forest 
(1600-1700 ma.s.l.) mixed with Ilex aquifolium and Quercus ilex, on calcareous 
soil (Malencon & Bertault 1975). It has recently been found in southern France 
(Bon 1987, Poumarat & Neville 1993) and in the Maurienne Valley (Charignon 
& Garcin 1998, Fourré 1997) in coniferous forests (Pinus sylvestris, Larix 
decidua, Picea abies) and always on calcareous soil. 

Clitocybe amoenolens has been responsible for several erythromelalgic-type 
poisonings in the Maurienne Valley (Savoie, France) (Moreau et al. 2001, Saviuc 
& Danel 2006). It also has been found in the Abruzzi region (Centre Italy) under 
Pinus nigra (Contu et al. 1999), P. nigra, and Cedrus spp. (Leonardi et al. 2002) 
and P. nigra and Larix decidua (Leonardi & Maggi 2007), and poisoning cases 
referable to C. amoenolens have been recognised in this region (Leonardi et al. 
2002, Marinetti & Recchia 2005). Finally, the species has been reported from 
Spain (Martinez et al. 2010), where it was collected in the autonomous regions 
of La Rioja (Picea abies) and Castilla-La Mancha (Pinus pinaster and Cupressus 
arizonica or P. nigra and Quercus petraea), close to the north and centre of 
Spain, respectively. 

Our collections are the first record from northern Italy. According to our 
observations and bibliographic data, this species seems strictly restricted to 
Pinaceae in higher altitude thermophilic forests on calcareous soils. 


Paralepistopsis gen, nov. and Paralepista ... 261 


Paralepistopsis acromelalga (Ichimura) Vizzini, comb. nov. 
MycoBank MB564342 
= Clitocybe acromelalga Ichimura, Bot. Gaz. (Tokyo) 65: 110 (1918). 

SELECTED DESCRIPTIONS — Ichimura (1918: 110); Moreau et al. (2001: 109-111). 

SELECTED ICONOGRAPHY — Guez (2010: (http://www.mycodb.fr/fiche.php?genre=Clit 

ocybe&espece=acromelalga ). 
HABITAT & DISTRIBUTION: Clitocybe acromelalga, described from Japan 
(Ichimura 1918, Imazeki & Hongo 1957, Imazeki et al. 1988; Romagnesi 1989, 
Guez 1990), also occurs in South Korea (Lee & Hong 1985). It was reported 
as growing on both angiosperm (Phyllostachys bambusoides, Acer palmatum, 
Zelkova serrata) and gymnosperm (Cryptomeria japonica) litter. 


Phylogeny and specific delimitation 

In our combined ITS-LSU phylogenetic tree (Fic. 1) Clitocybe amoenolens 
and its sister C. acromelalga are not closely related to C. nebularis (Batsch) 
P. Kumm., the type of the genus Clitocybe (Redhead et al. 2002), nor to other 
Clitocybe species or allied taxa. As these two species represent a new phyletic 
line of clitocyboid fungi, it seems most appropriate to transfer them to the new 
genus Paralepistopsis. 

Based on its habit, coloured lamellae and small spores, Bon (1997) and 
Moreau et al. (2001) placed C. amoenolens traditionally in subg. Clitocybe 
sect. Gilvaoideae Harmaja, where it occupies an isolated position. Contu et 
al. (1999), focusing on hymenial features (basidia longer than 30 um), placed 
it in subgen. Hygroclitocybe Bon sect. Clavipedes Harmaja, a subgenus shown 
in recent molecular analyses (Redhead et al. 2002, Vizzini et al. 2011) to be 
artificial and heterogeneous. 

The traditionally defined Lepista (Fr.) W.G. Sm. —clitocyboid fungi with 
a pinkish yellow spore print, usually separable lamellae, and inamyloid 
cyanophilous ornamented [verruculose to spiny] basidiospores (Singer 1986, 
Bon 1997, Consiglio & Contu 2003)— is a polyphyletic genus (Fic. 1). The 
species of Lepista subg. Paralepista (= Lepista sect. Gilva), which combine very 
crowded decurrent lamellae with subglobose to largely ellipsoidal spores, are 
not closely related either to Lepista s.s. or to other taxa in the tricholomatoid 
clade. Consequently we accept this lineage as a distinct genus and propose 
Paralepista for Lepista flaccida, L. gilva and allies. Following Bigelow (1985), 
Bon (1991), and Raithelhuber (2004), we list below all the taxa accepted in 
Paralepista: 


Paralepista Raithelh., Gattung Clitocybe 1: 17 (1981). 
TYPE SPECIES — Agaricus inversus Scop. 
= Lepista subg. Paralepista (Raitelh.) Bon, Doc. Mycol. 26(102): 18 (1996). 
= Clitocybe sect. Eulepistae Singer, Ann. Mycol. 41: 40 (1943). 


262 ... Vizzini & Ercole 


= Lepista sect. Gilva Harmaja, Karstenia 18: 53 (1978). 
“Lepista sect. Eulepista” Konrad & Maubl., Icon. Select. Fung. 6(10): 350 (1936), nom. inval. 


“Lepista sect. Inversae” Singer & Clémencon, Nova Hedwigia 
23: 310 (1973 [“1972”]), nom. inval. 


Paralepista abdita (Dérfelt) Vizzini, comb. nov. 
MycoBank MB 564343 
= Lepista abdita Dorfelt, Boletus 1(2): 37 (1997). 


Paralepista ameliae (Arcang.) Vizzini, comb. nov. 
MycoBAnk MB 564344 
= Clitocybe spinulosa var. ameliae Arcang., Nuovo Giorn. Bot. Ital. 21: 434 (1889). 


Paralepista biformis (Peck) Vizzini, comb. nov. 
MycoBANnkK MB 564345 
= Clitocybe biformis Peck, Bull. N.Y. St. Mus. 150: 25 (1911). 


Paralepista concentrica Raithelh., Metrodiana 23: 122 (1996). 


Paralepista femoralis (H.E. Bigelow) Vizzini, comb. nov. 
MycoBank MB 564346 
= Clitocybe femoralis H.E. Bigelow, Sydowia 36: 14 (1983). 


Paralepista flaccida (Sowerby) Vizzini, comb. nov. 
MycoBank MB 564347 
= Agaricus flaccidus Sowerby, Col. Fig. Engl. Fungi 2: pl. 185 (1799). 


Paralepista flaccida var. fibrillosa (Malencon) Vizzini, comb. nov. 
MycoBank MB 564348 
= Clitocybe flaccida var. fibrillosa Malencon, in Malencon & Bertault, 
Flore des champignons superieurs du Maroc 2 - Trav. Inst. 
Sci. Chérifien, Sér. Bot. Biol. Vég. 33: 157 (1975). 


Paralepista gilva (Pers.) Vizzini, comb. nov. 
MycoBANnkK MB 564349 
= Agaricus gilvus Pers., Syn. Meth. Fung.: 448 (1801). 
“Paralepista gilva” Raithelh., Metrodiana 23: 117 (1996), nom. inval. 


Paralepista inversa (Scop.) Raithelh., Gattung Clitocybe 1: 17 (1981). 
= Agaricus inversus Scop., Fl. Carniol., Ed. 2, 2: 445 (1772). 


Paralepista lentiginosa (Fr.) Vizzini, comb. nov. 
MycoBank MB 564350 
= Agaricus lentiginosus Fr., Epicr. Syst. Mycol.: 69 (1838). 


Paralepista maculosa (Sacc.) Vizzini, comb. nov. 
MycoBank MB 564352 
= Agaricus maculosus Peck, Bull. Buffalo Soc. Nat. Sci. 1: 
45 (1873), nom. illegit., non Pers. (1801). 
= Clitocybe maculosa Sacc., Syll. Fung. 5: 183 (1887). 


Paralepistopsis gen, nov. and Paralepista ... 263 


Paralepista pseudoparilis (Enderle & Contu) Vizzini, comb. nov. 
MycoBank MB 564353 
= Lepista pseudoparilis Enderle & Contu, Beitr. Kenn. Pilze Mittel. 13: 12 (2000). 


Paralepista repanda (Raithelh.) Raithelh., Metrodiana 23: 121 (1996). 
= Lepista repanda Raithelh., Metrodiana 14: 21 (1986 [“1985”]). 


Paralepista shafferi (H.E. Bigelow) Vizzini, comb. nov. 
MycoBank MB 564354 
= Clitocybe shafferi H.E. Bigelow, Beih. Nova Hedwigia 81: 339 (1985). 


Paralepista splendens (Pers.) Vizzini, comb. nov. 
MycoBank MB 564355 
= Agaricus splendens Pers., Syn. Meth. Fung.: 452 (1801). 


Discussion 

Paralepistopsis species are characterized by a habit (decurrent and crowded 
lamellae) and colours (ochre-orange tinges) reminiscent of Paralepista or 
Infundibulicybe Harmaja, a whitish to cream spore print, smooth cyanophilic 
spores often arranged in tetrads in dried specimens and rarely exceeding 
5(-6) um in length. Paralepista differs in having strongly ornamented spores 
(Raithelhuber 1995, 2004); Infundibulicybe is distinguished by smooth 
lacrymoid spores with confluent bases and cyanophobic spore walls (Harmaja 
2003). 

Paralepistopsis clusters with Cleistocybe Ammirati et al. and Catathelasma 
in the /catathelasma clade. Because of the low resolution and lack of BPP and 
MLB support within the tree, a more precise, accurate position for the new 
genus could not be suggested. The presence of decurrent lamellae, confluent 
pileus and stipe, pale reddish brown colouration, and growth on soil are 
characters shared by Paralepistopsis, Cleistocybe, and Catathelasma. Cleistocybe 
and Catathelasma are distinguished from Paralepistopsis mainly by a partial 
veil, divergent to interwoven hymenophoral trama, and larger cyanophobic 
spores (Ammirati et al. 2007, Vizzini 2009); in addition Catathelasma spores 
are amyloid (Singer 1986). 

Paralepistopsis amoenolens is delimited by a unique combination of macro- 
/micromorphological and chemical features, such as i) a strong aromatic, floral 
odour reminiscent of Tricholoma caligatum, Inocybe corydalina, Lepista irina, 
and Entoloma ameides caused by volatile metabolites identified by Fons et al. 
(2006) as methyl-(E)-cinnamate (also a key odorant of T. caligatum), methyl- 
benzoate, (E)-nerolidol, and methylanthranilate; ii) lamellae easily separating 
from the pileus context; iii) a cream coloured spore print; iv) smooth cyanophilic 
spores often arranged in tetrads; v) basidia reaching 35-40 um; vi) pileipellis 
hyphae with short diverticula; vii) abundant thromboplerous hyphae; viii) and 
the presence of the toxic metabolite, acromelic acid A, a powerful neurotoxic 


264 ... Vizzini & Ercole 


amino acid responsible for erythromelalgic poisoning and _ structurally 
homologous with kainic acid (a strong agonist of non-N-methyl-D-aspartate 
glutamate receptor subtypes) and domoic acid (Bessard et al. 2004). 
Paralepistopsis acromelalga differs from P. amoenolens morphologically in a 
darker pileus and stipe, a pileus that soon becomes depressed, more crowded 
lamellae, a different odour, thromboplerous hyphae occurring only rarely, 
smaller spores (Ichimura 1918, Romagnesi 1989, Guez 1990, Miyauchi 1998, 
Moreau et al. 2001), and a more complex metabolite pattern (presence of 
acromelic acids A—-E with 19 other toxins; Konno et al. 1983, 1988; Fushiya et 
al. 1990, 1992; Saviuc & Danel 2006). Additionally, our analyses show only a 
91% pairwise ITS sequence identity between P. acromelalga and P. amoenolens. 
Singer (1986) transferred P acromelalga to the heterogeneous genus Neoclitocybe 
Singer based on the presence of rare diverticulate hyphae in the pileipellis. 
Based on their small spores and the Paralepista-like habit, C. gilvaoides 
Kauffman and C. gracilis (H.E. Bigelow & A.H. Sm.) Harmaja (sect. Gilvaoideae) 
from the coniferous forests of North America and Scandinavia (Bigelow 1985, 
Harmaja 1969) may also belong to Paralepistopsis, but more recently collected 
specimens are needed to perform molecular and biochemical analyses. 


Acknowledgements 

We would like to thank Vladimir Antonin (Moravian Museum, Brno, Czech 
Republic), Joe Ammirati (University of Washington, Seattle, USA), and Shaun Pennycook 
(Auckland, New Zealand) for their pre-submission reviews. Our most sincere thanks 
are due to Marco Contu (Olbia, Italy) for helpful suggestions and comments. 


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MYCOTAXON 


http://dx.doi.org/10.5248/120.269 
Volume 120, pp. 269-276 April-June 2012 


Three new species of Septobasidium (Septobasidiaceae) 
from Hainan Province in China 


SUZHEN CHEN?” & LIN Guo'* 


'State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 
100101, China 
?Ocean University of China, Qingdao 266003, China 


* CORRESPONDENCE TO: guol@im.ac.cn 


AsstRact —Three new Septobasidium species are described: S. capparis on Capparis 
membranifolia associated with Andaspis sp., S. reevesiae on Reevesia longipetiolata associated 
with Lepidosaphes sp., and S. dacrydii on Dacrydium pierrei associated with Pinnaspis sp. 
They were collected from Hainan Province, China. 


Key worps —Pucciniomycetes, Septobasidiales, taxonomy 


To date, nine new species and three new Chinese records of Septobasidium 
have been discovered from Hainan Province (Chen & Guo 2011b,c,d, 2012, Lu 
& Guo 2009a, 2010b,c). An additional three new species of Septobasidium are 
reported as follows: 


Septobasidium capparis S.Z. Chen & L. Guo, sp. nov. Fics 1-6 
FUNGAL NAME FN570003 
Differs from Septobasidium euryae-groffii by its thicker hymenium and larger basidia. 
Type: China, Hainan Province, Changjiang, Baomeiling, alt. 250 m, on Capparis 


membranifolia Kurz (Capparaceae), associated with Andaspis sp. (Diaspididae), 
12.IV.2011, L. Guo 11599 (HMAS 263233, holotype). 


Erymo.ocy: The epithet refers to the substrate plant genus, Capparis. 


Basidiomata on branches, resupinate, 10-24 cm long, 1.5-4 cm wide, pale 
cinnamon-brown or brown; margin determinate, surface smooth, cracked at 
maturity. In section 1500-2000 um thick, composed of 3-8 layers. Subiculum 
brown, 40-70 um thick. Pillars brown, 80-100 um high, 70-130 um wide, 
branched outwards to form a hyphal layer, 500-550 um high. From hymenial 
layer the fungal hyphae renew growth successively to form hyphal layers and 


270 ... Chen & Guo 


Fic. 1. Septobasidium capparis (HMAS 263233, holotype). Basidia and basidiospores. 


hymenia. Hymenium brown, 100-170 um thick. Basidia arising directly from 
the hyphae without a probasidial cell, cylindrical, straight or slightly curved, 
4-celled, 45-56 x 8-12 um, hyaline or brown. Sterigmata conical, 6-8 x 3-5 
um, hyaline or brown. Basidiospores reniform, 16-20 x 4.5-8 um, hyaline. 
Haustoria consisting of irregularly coiled hyphae. 


ComMENTSs: Septobasidium capparis is similar to S. euryae-groffii C.X. Lu & 
L. Guo, which differs by its thinner hymenium (70-110 um) and smaller basidia 
(20-45 x 5-8 um; Lu & Guo 2010a). 


Septobasidium spp. nov. (China) ... 271 


Fics. 2-6 Septobasidium capparis (HMAS 263233, holotype). 2. Basidioma on branch. 
3-4. Basidia (arrows). 5. Haustoria. 6. Section of basidioma. 


272 ... Chen & Guo 


Fic. 7. Septobasidium reevesiae (HMAS 263427, holotype). Basidia and basidiospores. 


Septobasidium reevesiae S.Z. Chen & L. Guo, sp. nov. Figs 7-12 
FUNGAL NAME FN570004 
Differs from Septobasidium henningsii by its upright pillars and thicker stratified 
hymenial layer. 


Type: China, Hainan Province, Bawangling Natural Reserve, alt. 1030 m, on Reevesia 
longipetiolata Merr. & Chun (Sterculiaceae), associated with Lepidosaphes sp. (Diaspididae), 
25.X1.2010, Y.E. Zhu & FE. He 518 (HMAS 263427, holotype). 


Erymo.oey: The epithet refers to the substrate plant genus, Reevesia. 


Basidiomata on branches, resupinate, 7.5-8.5 cm long, 3-6 cm wide, cinnamon- 
brown; margin white, determinate, surface smooth, at first frequently forming 
orbiculate or discoideous patches, confluent in the old stage. The dense pillars 
are barely visible, especially near the margin. In section 1650-2200 um thick. 
Subiculum brown, 30-50 um thick. Pillars fasciculate, brown, 700-1170 um 
high, 40-100 um wide. Hymenial layer hyaline or brown, 730-1000 um thick, 
often forming 2-4 strata, with a brown, 2-7 um thick horizontal layer between 
the hymenia, and closely packed parallel upright hyphae. Basidia arising directly 
from the hyphae without a probasidial cell, cylindrical, straight or slightly 
curved, 4-celled, 37-55 x 8-13(-18) um, hyaline or brown; wall 1-2(-3) um 
thick. Sterigmata conical, 9-11(-23) x 3-4.5 um. Basidiospores reniform, 
15-16x5-6 um. Haustoria consisting of irregularly coiled hyphae. 


ComMENTs: Septobasidium reevesiae is similar to S. henningsii Pat., which differs 
by its thinner hymenial layer (60-400 um high) and characteristic slanting, 
entangled, anastomosing pillars (Couch 1938). 


Septobasidium spp. nov. (China) ... 273 


mn IT mt 


| 
‘ 
ba a as si Mook 


10 ym 


Fics. 8-12. Septobasidium reevesiae (HMAS 263427, holotype). 8. Basidiomata on branches. 
9. Stratified hymenial layer. 10. Basidia (arrow). 11. Haustoria. 12. Section of basidioma. 


274 ... Chen & Guo 


13 


Fic. 13. Septobasidium dacrydii (HMAS 263232, holotype). Basidia and basidiospores. 


Septobasidium dacrydii S.Z. Chen & L. Guo, sp. nov. Figs 13-18 
FUNGAL NAME FN570005 
Differs from Septobasidium apiculatum by its thicker section with pillars. 
Type: China, Hainan Province, Bawangling Natural Reserve, alt. 1050 m, on Dacrydium 


pierrei Hickel (Podocarpaceae), associated with Pinnaspis sp. (Diaspididae), 24.X1.2010, 
Y.F. Zhu & FE. He 529 (HMAS 263232, holotype). 


Erymo oey: The epithet refers to the substrate plant genus, Dacrydium. 


Basidiomata on branches, resupinate, 35 cm long, 1-6 cm wide, cinnamon- 
brown or dark brown; margin determinate, surface smooth, peeled off in the 
old stage, dark brown pillars and hyphal layers emerging. In section 1450-2000 
um thick. Subiculum brown, 45-70 um thick. From the subiculum forming 
pillars or hyphal layers. Pillars brown, higher or shorter, 850-1200 um high, 
35-110 um wide, or 100-200 um high, 40-80 um wide. Hyphal layer, 700-800 
uum high. Pillars and hyphal layers successively formed from the hyphal layer. 
Hymenium hyaline, 85-140 um thick. Basidia arising directly from the hyphae 
without a probasidial cell, cylindrical or clavate, straight or slightly curved, 
3-celled, 30-40 x 9-10 um, hyaline. Sterigmata conical, 10-15 x 4-5 um, 
hyaline. Basidiospores cylindrical or fusiform, 21-26x5-6.5 um, hyaline. 
Haustoria consisting of irregularly coiled hyphae. 

ComMENTs: Septobasidium dacrydii is similar to S. apiculatum Couch ex L.D. 
Gomez & Henk, which differs by its thinner section (250-550 um thick) and 
absence of pillars (Couch 1938). 


Including the three new species reported in this paper, 46 Septobasidium species 
have now been reported in China (Sawada 1933, Couch 1938, Teng 1963, Tai 


Septobasidium spp. nov. (China) ... 275 


14 15 


nny - 
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aad booed ha 


' 


ETT 
UATE 


mm 


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Fics. 14-18. Septobasidium dacrydii (HMAS 263232, holotype). 14. Basidioma on branch. 
15. Basidia (arrow). 16. A basidiospore. 17. Haustoria. 18. Section of basidioma. 


276 ... Chen & Guo 


1979, Kirschner & Chen 2007, Lu & Guo 2009a,b,c, 2010a,b,c, 2011, Lu et al. 
2010, Chen & Guo 201 1a,b,c,d, 2012). 


Acknowledgements 

The authors would like to express their deep thanks to Drs. Eric H.C. McKenzie 
(Auckland, New Zealand) and Shuanghui He (Beijing Forestry University) for serving 
as pre-submission reviewers, to Dr. Shaun Pennycook (Auckland, New Zealand) 
for nomenclatural review, to Prof. Zhenyu Li (Institute of Botany, Chinese Academy 
of Sciences) and Mr. Qing Chen (Bawangling Natural Reserve, Hainan Province) 
for identifying the host plants, to Prof. Sanan Wu (Beijing Forestry University) for 
identifying the scale insects, and to Mrs. Xiangfei Zhu for inking in line drawings. This 
study was supported by the foundation of Ministry of Science and Technology of the 
People’s Republic of China (No. 2006FY110500-5). 


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109: 103-106. http://dx.doi.org/10.5248/109.103 

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Lu CX, Guo L. 2009c. Septobasidium annulatum sp. nov. (Septobasidiaceae) and Septobasidium 
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ISSN (print) 0093-4666 © 2012. Mycotaxon, Ltd. ISSN (online) 2154-8889 


MY COTAXON 


http://dx.doi.org/10.5248/120.277 
Volume 120, pp. 277-286 April-June 2012 


Characterization of the causal agent of poplar anthracnose 
occurring in the Beijing region 


ZHENG Lr, YING MEI LIANG? & CHENG MING TIAN” 


'Key Laboratory for Silviculture and Conservation of Ministry of Education, 
Beijing Forestry University & ?Museum of Beijing Forestry University, 
Beijing 100083, P. R. China 

* CORRESPONDENCE TO: chengmt@bjfu.edu.cn 


ABSTRACT — Twenty fungal isolates derived from infected poplar leaves collected in four 
Beijing regional districts during 2009 and 2010 were examined for morphological and cultural 
characteristics. Multi-gene phylogenetic sequence analyses of the rDNA ITS (ITS1-5.8S-ITS2) 
region and the B-tubulin2, partial actin (ACT), glyceraldehyde-3-phosphate dehydrogenase 
(GPDH), and glutamine synthetase (GS) genes were performed for these isolates. The 
morphological and cultural evaluations and DNA sequence analyses demonstrated that 16 
isolates represented Colletotrichum gloeosporioides while the remaining four isolates, all from 
Shi Jingshan district, represent a new species, Colletotrichum populi, which is described and 
illustrated. 


KEY worps — disease, multi-locus phylogeny, taxonomy 


Introduction 

The genus Colletotrichum causes anthracnose disease on a wide range of 
plants worldwide. The main Colletotrichum species causing anthracnose of 
forest trees include C. gloeosporioides, C. acutatum, and C. crassipes, which 
may cause infections of leaves and fruits of plants such as poplar, China fir, 
and paulownia, often leading to premature defoliation (Xu et al. 2004). Of 
these, C. gloeosporioides is the most common, affecting a wide range of tree 
hosts. The taxonomy of C. gloeosporioides is extremely complex because of its 
heterogeneous nature and instability in cultural characteristics. For example, 
more than 600 C. gloeosporioides synonyms have been identified (von Arx 
1957). Different experimental conditions and measurement errors may have 
a marked influence over the recorded sizes of conidia. Identification results 
obtained through traditional morphological examination are thought to be 
unreliable (Cai et al. 2009) when compared to molecular methods, and several 
new species, such as C. asianum and C. cordylinicola (Prihastuti et al. 2009, 


278 ... Li, Tian & Liang 


TABLE 1. Isolates of Colletotrichum used in study. 


ISOLATE No. Host ORIGIN SAMPLING DATE 
1C5-7 Populus xbeijingensis Chang ping 2010.08 
C1-5-1 Populus xbeijingensis Chang ping 2010.08 
2C5-8 Populus xbeijingensis Chang ping 2010.08 
25-9 Populus xbeijingensis Chang ping 2010.08 
2C5-14 Populus xbeijingensis Chang ping 2010.08 
3C5-3 Populus xbeijingensis Chang ping 2010.08 
4C5-2 Populus xbeijingensis Chang ping 2010.08 
C2-5 Populus xbeijingensis Chang ping 2010.07 
Y3-4 Populus cathayana Yan qing 2010.08 
Y3-6 Populus cathayana Yan qing 2010.08 
Yp1-7 Populus cathayana Yan qing 2010.08 
Bh7-2 Populus xbeijingensis Yan qing 2010.09 
Bh9-1 Populus cathayana Yan qing 2010.09 
Bh12-2 Populus xbeijingensis Yan qing 2010.09 
Dy10-2 Populus nigra Yan qing 2010.09 
HMBFU191 Populus nigra var. italica Shi Jingshan 2009.10 
HMBFU163 Populus nigra var. italica Shi Jingshan 2009.10 
HMBFU173 Populus nigra var. italica Shi Jingshan 2009.10 
HMBFU141 Populus nigra var. italica Shi Jingshan 2009.10 
M1-6 Populus nigra Mi yun 2010.08 


Phoulivong et al, 2010a), have recently been separated from C. gloeosporioides 
based on the results from molecular analysis. 

Colletotrichum gloeosporioides (teleomorph Glomerella cingulata) has been 
reported as the main pathogen of poplar anthracnose in China and North 
America (He et al. 1990, Newcombe 2000). Colletotrichum graminicola, which 
usually infects monocotyledonous plants such as cereals or grasses, may also 
infect poplar in India (Dipak et al. 1999). Most studies that are based on 
morphology may be contradicted by molecular data (Phoulivong et al. 2010b). 

The present study was carried out to determine the Colletotrichum species 
associated with poplar anthracnose in Beijing area through morphological 
examination and molecular methods. 


Materials & methods 


Isolation of Colletotrichum species 

Twenty Colletotrichum isolates were obtained from lesions on infected leaves of 
different poplar species collected in four districts in Beijing: Chang ping, Yan qing, 
Mi yun, and Shi Jingshan (TaBLeE 1). These isolates are deposited at the Mycological 
Herbarium of Beijing Forestry University (BJFC). 

Three 5x5 mm samples cut from the interface of healthy and diseased tissue 
were surface sterilized by dipping in 70% ethanol for 1 minute, then in 1% sodium 
hypochlorite for 3-4 minutes and washing in three changes of sterile water. The leaf 
segments were then placed onto the surface of potato dextrose agar (PDA) and incubated 


Colletotrichum populi sp. nov. (China) ... 279 


at room temperature (28°C). The growing edges of fungal hyphae developing from the 
leaf segments were transferred onto a new PDA plate after 1-2 days. Each isolate was 
purified through single spore isolation and was maintained on a new PDA plate at 28°C 
for further study. 


Morphological studies 

Mycelial discs (5 mm diam) derived from the edge of a 7-day old culture were 
transferred onto PDA and incubated at 28°C in the dark. Each colony radius was 
measured daily after the 2nd day for 4 days to determine the growth rate by averaging 
the daily growth (cm per day) of the colonies. Colony characters were also recorded 
and described. After 7 days a spore suspension (5x10° conidia/ml) was made from each 
culture and the size and shape of 30 conidia were measured by light microscopy at a 
magnification of 40x. A drop of the spore suspension was placed on a slide, which was 
placed in a Petri dish containing water-soaked filter paper to keep moist. After 24 hours 
(at 28°C) conidial germination was observed, and the size and shape of 20 appressoria 
were measured. 

Statistical data (morphology and growth rate) were analyzed using SPSS software 
version 11.5.0 (SPSS Inc., USA). 


DNA extraction and PCR amplification 

Mycelium was scraped from the surface of 7-day old cultures of each isolate and dried 
using sterilized filter paper. Genomic DNA was extracted using the modified CTAB 
method. Extracted DNA was electrophoresed on 1% agarose gel to check genomic DNA 
quality. 

Using polymerase chain reaction (PCR), DNA was amplified from the complete 
rDNA-ITS (ITS) region and the $-tubulin (TUB2), partial actin (ACT), glutamine 
synthetase (GS), and glyceraldehyde-3-phosphate dehydrogenase (GPDH) genes of 
each Colletotrichum isolate. The primer pairs for the amplified genes are ITS1+ITS4 
(White et al. 1990), Bt2a+Bt2b (Glass et al. 1995), ACTF+ACTR (Carbone & Kohn 
1999), GSF1+GSR1 (Guerber et al. 2003), GDF1+GDRI1 (Peres et al. 2008) respectively. 
Amplification was performed in 25 wL volume with 1 uL (20 ng) DNA template, 2 uL 
dNTP (2.5 mmol/L), 2.5 uL 10xPCR buffer, 0.5 uL of each primer (10 uLmol/L), 0.15 uL 
Taq DNA polymerase and 18.35 uL ddH,O. The PCR profile consisted of denaturation at 
95°C for 3 min, followed by 34 cycles at 95°C for 1 min, 55°C for 30 s, 72°C for 1 min and 
a final cycle at 72°C for 10 min. PCR products were electrophoresed on 1% agarose gel 
and DNA sequencing was carried out by the Shanghai Invitrogen Biological Technology 
Co. Errors were adjusted manually with BioEdit software version 7.0.5.3 (© Tom Hall). 
All isolate and DNA sequence accession (DDBJ, EMBL, GenBank) numbers are listed 
in TABLE 2. 


Phylogenetic analysis 

The sequences of Colletotrichum isolates were aligned using Clustal X (1.81). 
Alignments were manually adjusted to allow maximum alignment and maximum 
sequence similarity, and gaps were treated as missing data. 

For parsimony analysis, PAUP version 4.0b10 was used and heuristic search was 
performed with 1000 repeats of random addition sequences in Stepwise-Addition 
Option and TBR swapping algorithm in Branch Swapping Option. Confidence limits 


280 ... Li, Tian & Liang 


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Colletotrichum populi sp. nov. (China) ... 281 


for the branches based on parsimony criteria were estimated by bootstrap analysis of 
1000 replicates. Phylogenetic trees were constructed from distance matrix values by 
the maximum parsimony (MP) method. Describing trees was based on tree length, 
consistency index (CI), retention index (RI), rescaled consistency index (RC) and 
homoplasy index (HI). Constructed trees were viewed by TreeView software. The 
GenBank accession numbers used as references in this study are listed in TABLE 2. 


Results 


Morphological and cultural characterization 

The isolates could be divided into two groups (TABLE 3) based on differences 
in cultural characteristics and appressorial morphology. Group 1 consisted of 
16 isolates with colonies varying from white or grey to dark brown and turning 
dark grey or olive-green with time; aerial mycelium white or grey (PLATE 
1a-B). These cultures produced abundant orange conidial masses around the 
inoculation point. The conidia were cylindrical, obtuse at both ends or slightly 
tapered at one end, possessing 2-3 vacuoles and measuring 9.8-22.0 x 2.9-6.9 
um (PLATE lc). Appressoria were generally round or ovate and 4.9-15.9 x 
4.4-9.8 um (PLATE ID). 


TABLE 3. Morphological and cultural characteristics of 20 Colletotrichum isolates. * 


C. gloeosporioides C. populi 
(Group 1) (Group 2) 
ISOLATES TESTED 16 4 
CONIDIA 
SHAPE Cylindrical Cylindrical 
LENGTH (um) 15.8 + 1.9 (9.8-22.0) 15.1 + 2.0 (9.8-17.7) 
WIDTH (um) 4.9 + 0.4 (2.9-6.9) 5.2 + 0.9 (3.0-7.3) 
APPRESSORIA 
SHAPE Circular or ovate Irregular, edge lobed 
LENGTH (um) 9.44 2.1 (4.9-15.9) 9.4 + 1.6 (7.3-12.2) 
WIDTH (um) 5.9 + 1.0 (4.4-9.8) 6.2 + 1.2 (4.9-9.3) 
COLONY Light, dark grey or brown; Aerial mycelium cottony, 
aerial mycelium grey white, 
with orange conidial masses with indistinct conidial masses 
DaILy GROWTH (cm/d) 0.72 0.08 (0.56-0.88) 0.73 0.05 (0.62—0.80) 


* Measurements are presented as mean + sd (range). 


Group 2 consisted of four isolates collected from Shi Jingshan district. The 
colonies were white to light grey in color and covered with white cottony aerial 
mycelia, with indistinct conidia masses on the surface of colonies. Conidia 
were similar to those of Group 1 in shape (cylindrical) and size (9.8-17.7 x 
3.0-7.3 um), but had 1-2 vacuoles. Appressoria were irregular in shape with 
lobed edges and measured 7.3-12.2 x 4.9-9.3 um. 

There were no significant differences in growth rate between the two groups, 
both showing an average daily growth of ca. 0.72 cm/d. 


282 ... Li, Tian & Liang 


Multi-locus phylogeny 

The sequence information obtained from five genes and the phylogenetic 
tree information based on each gene sequence are shown in TABLE 4. Of the 
five genes, GS and GPDH had the highest rates of parsimony informative 
characters (PIC), GS comprising 439 (40.1%) PIC out of a total 1095 characters 
and GPDH 114 (38.6%) PIC out of 295 characters. 

The phylogenetic tree was constructed on the basis of combined data of the 
five genes (PLATE 2). The best tree contained 2593 characters with NPIC=681, 
CI=0.865, RI=0.853, HI=0.135, RC=0.738 and Length=1712, showing that the 
two morphologically distinct groups were placed into two separate branches. 
Group 1 clustered with C. gloeosporioides with a bootstrap value of 100%. 
Group 2 was distinctive from C. fructicola (bootstrap value = 79%) and other 
members in ‘gloeosporioides complex’ (bootstrap values > 90%), suggesting the 
probability of a new species. 


TABLE 4. Gene regions amplified. 


DNA SEQUENCE LENGTHS PHYLOGENETIC TREE 
bee (BP) NPIC/CHARACTERS (%) 
ITS 534-537 8.0 
TUB2 440-441 18.4 
ACT 241 20.1 
GPDH 221-230 38.6 


GS 981-988 40.1 


Taxonomy 


Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. PLATE 1 

Lesions on leaves of poplar as black spots distributed randomly or in 
circles with a dark brown border, orange conidial masses sometimes observed. 
Conidia cylindrical, 9.8-22.0 x 2.9-6.9 um, obtuse at ends, hyaline, smooth. 
Appressoria ovate or circular, 4.9-15.9 x 4.4-9.8 um. Colonies on PDA with 
white, grey, dark grey, or olive grey mycelium and orange sporulation. Reverse 
brown or olive-green with dark spots. 


PiaTE 1. Colletotrichum gloeosporioides isolates (Group 1): cultural and morphological characters. 
A: Colony of isolate 2C5-14; B: Colony of isolate BH7-2; C: Conidia of isolate M1-6; D: Clavate or 
circular appressoria of isolate C1-5-1. Bars = 10 um. 


Colletotrichum populi sp. nov. (China) ... 283 


Group | 


2C5-14 
BH12-2 


Colletotrichum populi HMBFU163 

Colletotrichum ee HMBFU191 Group 2 
Colletotrichum populi HMBFU173 
Colletotrichum populi HMBFU141 
Colletotrichum fructicola BPD-I12 
Colletotrichum siamense BML-115 
Colletotrichum jasmine-sambac LLTA-01 
Colletotrichum asianum BPD-14 
Colletotrichum kahawae IMI319418 
Colletotrichum cordylinicola LC0856 
olletotrichum horii TSG002 
Colletotrichum acutatum CBS294.67 
Colletotrichum simmondsii BRIP28519 


(New Species) 


Colletotrichum boninense C05016 


_10 


PLaTE 2. Phylogenetic tree based on the combination of sequences of ITS, TUB2, ACT, GPDH, 
and GS genes (MP.) 


Colletotrichum populi C.M. Tian & Zheng Li, sp. nov. PLATE 3 
MYCOBANK 564436 


Lesions with black spots in circle and a light brown border, sometimes sunken. Differs 
from Colletotrichum fructicola by grey-white PDA colonies (reverse grey white or pale 
yellow) and from C. gloeosporioides by longer and irregularly lobed appressoria formed 
from conidia. 


Type: China. Beijing, Shi Jingshan District, from leaves of Populus nigra var. italica, 25th 
October 2009, coll. C.M. Tian, HMBFU191 (Holotype BJFC, lyophilized PDA culture). 


ETyMo_ocy: populi, referring to the host species. 


On leaves of poplar producing circular black spots with a dark brown border. 
Colonies on PDA at first white, becoming grey-white, reverse grey white or pale 
yellow, in 5 days at 28°C, daily growth 0.62-0.80 cm/day (mean = 0.73 + 0.05, 
n=5). Aerial mycelium cottony, white with few conidial masses, setae absent in 
culture. Conidia 9.8-17.7 x 3.0-7.3 um (mean = 15.1 + 2.0 x 5.2 + 0.9, n = 30), 
conidia smooth-walled, hyaline, and cylindrical with obtuse ends. Appressoria 
7.3-12.2 x 4.9-9.3 um (mean = 9.4 + 1.6 x 6.2 + 1.2, n = 20), formed from 
conidia, brown to dark brown, irregularly lobed in shape. 


ADDITIONAL COLLECTIONS EXAMINED: CHINA. BEING, Shi Jingshan District, from 
leaves of Populus nigra var. italica, 25th October 2009, coll. C.M. Tian, HMBFU161, 
HMBFU141, HMBFU173 (BJEFC, lyophilized PDA cultures). 


284 ... Li, Tian & Liang 


aay 


| 
rn | 
ih = 


PLaTE 3. Colletotrichum populi isolates (Group 2): cultural and morphological characters of 
holotype (isolate 1-9-1). A: Colony; B: Conidia; C & D: irregular appressoria. Bars = 10 um. 


ComMENTs: Colletotrichum populi can be separated from the other species 
by both morphological and molecular characters (TABLE 5). The similar 
C. asianum grows more slowly and produces narrower conidia (8.7-20.3 
x 3-4.7 um). The new species shares similar colony characteristics with 
C. boninense, C. gloeosporioides, and C. kahawae, all of which produce shorter 
and more rarely lobed appressoria. The genetically most closely related C. fructi- 
cola is distinguished by colonies that are grayish green in reverse with white 
edges. Colonies of C. hymenocallidis, which has similarly sized conidia and 
appressoria, appear denser in the central zone and produce fusiform conidia. 
C. jasmine-sambac differs by its colony with fimbriate margin. 


Discussion 

Both Colletotrichum gloeosporioides and C. populi have been found to cause 
anthracnose on poplars in Beijing region but can be distinguished by differing 
colony and appressoria morphologies as well as by molecular data derived from 
sequencing five genes. Appressorial size and shape have been used to distinguish 
some Colletotrichum species. For example, C. gloeosporioides was distinguished 
from C. acutatum by the shape of appressoria (Du et al. 2005). Crouch & Beirn 
(2009) pointed out that the size and shape of appressoria might be effective 
with the combination of host species during their work on anthracnose of 
cereals and grasses 

Although rDNA-ITS gene is not an ideal marker for intraspecific relation- 
ships, it is very useful in many cases for reconstruction of interspecific 
relationships (Cai et al. 2009). Other gene sequences have been examined in 
the phylogenetic analysis of Colletotrichum. GS and calmodulin (CAL) were 
found to be useful when studying anthracnose on coffee berries (Prihastuti et 
al. 2009). Compared with six gene regions, GPDH, CAL and ACT were useful 
for inferring the relationship of C. gloeosporioides sensu lato (Cai et al. 2009). 
Whilst there is no defined set of genes to be used for taxonomic studies of 
Colletotrichum, molecular data from a wider range of genes would give better 
insights into this group of fungi. 


TABLE 5. Colletotrichum populi and closely related species. 


Colletotrichum populi sp. nov. (China) ... 285 


SPECIES COLONY ConrDIA (tm) eee conc REFERENCE 
Gandini Be Pte Cyl. with obtuse ends, eae ish Prihastuti 
HieMlascen 8.7-20.3 x 3-4.7 gas elin ad et al. 2009 
White aerial 
mycelium Irregularly Jouji et al 
C. boninense y { Cyl, 11.5-17 x 4-7 shaped, J : 
reverse cream to 2003 
6-17 x 4-15 
orange 
: Ovoid, occ. 
: White; in age grey Ca lawn ene ObuISe te clavate, oft Prihastuti 
C. fructicola sl. rounded, 
at the center 85-16 x 3.5-5 complex, et al. 2009 
6-9 x 5.5-7 
a Coes wine ee Cyl. base truncate, Glare Sutton 
C. gloeosporioides dark grey, aerial obovate, 
4 12-17 x 3.5-6 1992 
mycelium 6-20 x 4-12 
cs White; later pale Fusiform, straight, ends Ovate, occ. Yang et al. 
C. hymenocallidis a ih obtuse clavate, 
grey with circles 14-20 x 5-6.5 711% 5-75 2009 
White aerial Cyl, straight with Shape variable, : 
ow ; . ; : Wikee et al. 
C. jasmine-sambac —_ mycelium, with obtuse ends, hyaline, ovoid, clavate, sl. 2010 
fimbriate margin 13-15 x 3.5-4 irregular, brown 
F : Cyl. straight with Irregular, edge F 
a erie cae pbnuse-cnds, eee nes 
¥ 9.8-22.0 x 2.9-7.3 7.3-12.2 x 4.9-9.3 
Grey erences Straight, cyl., guttulate, Circular to sl. Piihaetuti 
C. kahawae barker alse: are apex obtuse, irregular, et al. 2009 
PON ee TGA TRS BS 4.5-10 x 7.5 


Acknowledgments 


We wish to thank Professor M.H. Pei and Dr. C.J. You for text amendments. We are 
also grateful to Professor Q. Lu (Chinese Academy of Forestry Sciences, Beijing) and Dr 
E.H.C. McKenzie (Landcare Research, Auckland, New Zealand) for their constructive 
reviews and valuable suggestions. This research was funded by Forestry Commonweal 
Industry Scientific Research Plan (no. 201004003). 


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Colletotrichum gloeosporioides is not a common pathogen on tropical fruits. Fungal Diversity 
44: 33-43. http://dx.doi.org/10.1007/s13225-010-0046-0 

Phoulivong S, Cai L, Parinn N, Chen H, Abd-Elsalam K, Chukeatirote E, Hyde KD. 2010b. A new 
species of Colletotrichum from Cordyline fruticosa and Eugenia javanica causing anthracnose 
disease. Mycotaxon 114: 247-257. http://dx.doi.org/10.5248/114.247 

Prihastuti H, Cai L, Chen H, McKenzie EHC, Hyde KD. 2009. Characterization of Colletotrichum 
species associated with coffee berries in Chiang Mai, Thailand. Fungal Diversity 39: 89-109. 

Sutton BC. 1992. The genus Glomerella and its anamorph Colletotrichum. 1-26, in: JA Bailey et al. 
(eds). Colletotrichum - Biology, Pathology and Control, CAB International. 

White TJ, Bruns T, Lee S, Taylor JW. 1990. Amplification and direct sequencing of fungal ribosomal 
RNA genes for phylogenetics. 315-322, in: MA Innis et al. (eds). PCR Protocols: a guide to 
methods and application. San Diego, Academic Press. 

Wikee S, Cai L, Pairin N, McKenzie EHC, Su YY, Chukeatirote E, Thi HN, Bahkali AH, Moslem 
MA, Abdelsalam K, Hyde KD. 2011. Colletotrichum species from jasmine (Jasminum sambac). 
Fungal Diversity 46: 171-182. http://dx.doi.org/10.1007/s13225-010-0049-x 

Xu HM, Chen JY, Xiao DL. 2004. Progress in study on anthracnose from forest tree. Hubei Forestry 
Science and Technology 130: 40-42. 

Yang YL, Liu ZY, Cai L, Hyde KD, Yu ZN, McKenzie EHC. 2009. Colletotrichum anthracnose of 
Amaryllidaceae. Fungal Diversity 39: 123-146. 


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MY COTAXON 


http://dx.doi.org/10.5248/120.287 
Volume 120, pp. 287-290 April-June 2012 


Sympodioplanus yunnanensis, a new aquatic species 
from submerged decaying leaves 


GUANG-ZHU YANG, Kat-PING Lu, YUE YANG, LI-Bo Ma, 
MIN QIAO, KE-QIN ZHANG & ZE-FEN Yu* 


‘Laboratory for Conservation and Utilization of Bio-resources, 
Key Laboratory for Microbial Resources of the Ministry of Education, 
Yunnan University, Kunming, Yunnan, 650091, PR. R. China 
*CORRESPONDENCE TO: zfyuqm@hotmail.com 


ABSTRACT — Sympodioplanus yunnanensis sp. nov. from submerged leaves collected from 
Gengma county, Lincang city, was found when researching aquatic hyphomycetes in Yunnan. 
The new species is well defined as a Sympodioplanus by its sympodial proliferation with 
multiple and crowded conidiogenous loci in the upper conidiophores and elongated conidia. 
It differs in conidial shape and septation from the type species, S. capensis. 


KEY worpDs — anamorphic fungi, taxonomy 


Introduction 

Fungal diversity in southern China is high, and many anamorphic fungi 
collected in Yunnan Province have been published (Ma & Zhang 2007a,b,c, 
Shang & Zhang 2007, Wang & Zhang 2007, Ma et al. 2008, 2010, 2011, Zhang 
et al. 2009a,b,c, 2011a,b). 

Sinclair et al. (1997) erected Sympodioplanus R.C. Sinclair & Boshoff for 
anamorphic fungi that exhibit sympodial proliferation with multiple and 
crowded conidiogenous loci in the upper part of the brown conidiophore, and 
elongated conidia with septa. Up to now, the monotypic genus comprised only 
S. capensis R.C. Sinclair & Boshoff isolated from dead decorticated wood. We 
add here a second species, S. yunnanensis, isolated from submerged decaying 
leaves. 


Materials & methods 

In 2010, a culture was isolated from leaves of a dicotyledonous plant submerged in 
a river in Yunnan Province, China. A 2 x 5 cm rotten leaf was spread on the surface 
of CMA (20 g cornmeal, 18 g agar, 40 mg streptomycin, 30 mg ampicillin, 1000 mL 
distilled water) and incubated for ten days; single conidia were isolated using a sterilized 


288 ... Yang & al. 


A 


PLATE 1. Sympodioplanus yunnanensis (holotype: YMF1.03797). A-F. Conidia. G. Conidiophores 
bearing conidia. H-I. Conidiophores showing moderate protuberance in the apex region. 
Scale bars: A-F = 10 um, G =25 um, H-I = 10um. 


Sympodioplanus yunnanensis sp. nov. (China) ... 289 


toothpick viewed under a CX31 microscope and cultivated on CMA in Petri plates. 
Morphological observations were made from CMA after incubation at 25°C for one week; 
pure cultures and a permanent slide were deposited in the herbarium of Laboratory for 
Conservation and Utilization of Bio-resources, Yunnan University, Kunming, Yunnan, 
PR. China (YMF). 


Taxonomy 


Sympodioplanus yunnanensis G.Z. Yang & Z.F. Yu, sp. nov. PLATE 1 
MycoBank MB 563548 
Differs from Sympodioplanus capensis by its larger conidia with more numerous septa. 


Type: PR China, Yunnan Province, Lincang city, Meihua mountain, 23°38'N 99°22’E, 
elev. 1925 m, in a river on submerged leaves of an unidentified dicotyledonous plant, 
Dec 2010, G.Z. Yang (Holotype: YMF 1.03797; ex-type culture YMF 1.03797). 


ETYMOLOGY: yunnanensis refers to the province in which the species was found. 


Colonies effuse, brown, sparse, hairy, often inconspicuous, attaining 30 mm 
diam after 10 days on CMA at 25°C. Mycelium mostly immersed in the 
substrate, composed of branched, septate, hyaline to brown, smooth, 1.0-3.0 
uum wide hyphae. Conidiophores macronematous, mononematous, solitary 
or in groups, erect, simple, straight, septate, brown, paler towards the apex, 
smooth, < 70-205 um long, 4.5-6.7 um wide. Conidiogenous cells holoblastic, 
polyblastic, integrated and terminal, sympodial, thin-walled, bearing multiple, 
closely approximated protuberances at the upper cells, including the apex. Scars 
are flat in relation to the adjacent conidiogenous cell wall and not thickened, 
0.5-1.0 um wide. Conidia solitary, smooth, mostly spindle with truncate base, 
occasionally constricted at septa, at the apex mostly slightly attenuate, 38-56 x 
6.0-10.8 um, thin-walled, mostly 7-, sometimes 6-, 5-, or 4-septate. 


ComMENTs: Sympodioplanus yunnanensis was easily determined as a 
Sympodioplanus by its similarity to the type species (S. capensis) in exhibiting 
sympodial proliferation with closely placed multiple conidiogenous loci in the 
upper conidiophore that are flat and equally thick as the conidiophore wall, 
forming solitary terminal and lateral conidia with cell walls similar to those 
of the upper conidiophore, and in producing elongated conidia with eusepta. 
However, the conidia of S. capensis are smaller (13-16(-19) x 2.5-4 um) and 
have fewer septa (3(-5)-septate). 


Acknowledgements 

This work was jointly financed by National Natural Science Foundation Program 
of PR China (31160008, 31060008), Grants from the Young Academic and Technical 
Leader Raising Foundation of Yunnan Province (2010CI020). We are very grateful to 
Drs. X.G. Zhang and H.-O. Baral for critically reviewing the manuscript and providing 
helpful suggestions to improve this paper. 


290 ... Yang & al. 


Literature cited 

Ma J, Zhang XG. 2007a. Three new species of Corynespora from China. Mycotaxon 99: 353-358. 

Ma J, Zhang XG. 2007b. Taxonomic studies of Sporidesmium from China. Mycotaxon 99: 
367-371. 

Ma J, Zhang XG. 2007c. Two new species of Sporidesmium from Yunnan, China. Mycotaxon 101: 
73-76. 

Ma J, Zhang K, Zhang XG. 2008. Two new species of the genus Minimelanolocus in China. 
Mycotaxon 104: 147-151. 

Ma IG, Ma J, Zhang YD, Zhang XG. 2010. A new species of Spadicoides from Yunnan, China. 
Mycotaxon 113: 255-258. http://dx.doi.org/10.5248/113.255 

Ma LG, Ma J, Zhang YD, Zhang XG. 2011. Taxonomic studies of Endophragmiella from southern 
China. Mycotaxon 117: 279-285. http://dx.doi.org/10.5248/117.279 

Shang ZQ, Zhang XG. 2007. Two new Corynespora species from Jiangsu, China. Mycotaxon 100: 
155-158. 

Sinclair RC, Boshoff S, Eicher A. 1997. Sympodioplanus, a new anamorph genus from South Africa. 
Mycotaxon 64: 365-374. 

Wang XM, Zhang XG. 2007. A new species of Corynespora from Yunnan, China. Mycotaxon 101: 
79-81. 

Zhang K, Fu HB, Zhang XG. 2009a. Taxonomic studies of Minimelanolocus from Yunnan, China. 
Mycotaxon 109: 95-101. http://dx.doi.org/10.5248/109.95 

Zhang K, Ma LG, Zhang XG. 2009b. New species and records of Shrungabeeja from southern 
China. Mycologia 101: 573-578. http://dx.doi.org/10.3852/09-006 

Zhang K, Ma J, Wang Y, Zhang XG. 2009c. Three new species of Piricaudiopsis from southern 
China. Mycologia 101: 417-422. http://dx.doi.org/10.3852/08-147 

Zhang YD, Ma J, Ma LG, Zhang XG. 2011a. Two new species of Taeniolina from southern China. 
Mycological Progress 11: 71-74. http://dx.doi.org/10.1007/s11557-010-0729-7 

Zhang YD, Ma J, Wang Y, Ma LG, Castafieda-Ruiz RF, Zhang XG. 2011b. New species and 
record of Pseudoacrodictys from southern China. Mycological Progress 10: 261-265. 
http://dx.doi.org/10.1007/s11557-010-0696-z 


ISSN (print) 0093-4666 © 2012. Mycotaxon, Ltd. ISSN (online) 2154-8889 


MY COTAXON 


http://dx.doi.org/10.5248/120.291 
Volume 120, pp. 291-294 April-June 2012 


Phaeotrichoconis crotalariae, 
endophytic on Vitis labrusca in Brazil 


THAIS EMANUELLE FEIJO DE LIMA”, JOSE LUIZ BEZERRA 
& MARIA AUXILIADORA DE QUEIROZ CAVALCANTI* 
Departamento de Micologia, Universidade Federal de Pernambuco, 


Rua Nelson Chaves, s/n, Cidade Universitaria, Recife, 50670-901, Brazil 
* CORRESPONDENCE TO: thaisfeijo@gmail.com, xiliamac@gmail.com 


ABSTRACT — Phaeotrichoconis crotalariae was isolated during studies with endophytic fungi 
on healthy leaves of Vitis labrusca in Pernambuco, Brazil. This is a new host for P. crotalariae, 
and the second report of this fungus from Brazil. 


KEY worps — Dematiaceae, Vitaceae, taxonomy 


Introduction 

The genus Phaeotrichoconis was proposed by Subramanian (1956) after 
Salam & Rao (1954) reassessed Trichoconis crotalariae originally described on 
leaves of Crotalaria verrucosa L. The new genus was described as an anamorphic 
dematiaceous fungus with phragmosporic acrogenous brown elongated 
fusiform conidia with a long appendix. 

Phaeotrichoconis crotalariae has a pantropical distribution in Africa, Asia, 
Australia, North America, and South America. It is reported to cause foliar flecks 
on Acacia spp. and has also been recorded on Alternanthera, Chrysalidocarpus, 
Cocos, Crotalaria, Cyperus, Elaeis, Marsilea, Oryza, Rotala, and Xanthium 
(Chase 1982, Ellis 1971, Miller 1997, Mishra et al. 1972, Mohanan & Sharma 
1988, Old et al. 1996, Ramakrishnan et al. 1972). In Brazil, it has been reported 
from Ceara State as a leaf endophyte of Copernicia prunifera (Freire & Bezerra 
2001). 

Phaeotrichoconis crotalariae was found in healthy leaves of Vitis labrusca 
in the Siriji Valley (municipalities of Sao Vicente Férrer and Macaparana), 
Pernambuco, Brazil. This paper presents a description of the fungus in culture. 


292 ... Lima, Bezerra & Cavalcanti 


Pate 1. Phaeotrichoconis crotalariae. 
A. sclerotia; B. conidiophore; C-D. young conidia; E-H. mature conidia. 


Phaeotrichoconis crotalariae endophytic on Vitis ... 293 


Materials & methods 

During the February 2010 dry season, healthy mature leaves of Vitis labrusca were 
collected from forest areas of the municipalities of Sao Vicente Férrer and Macaparana, 
Pernambuco. 

In the laboratory, each leaf was washed gently in running water with soap. Leaf discs 
were cut with a sterile metallic cork punch (6 mm diam.), decontaminated with 70% 
alcohol for 30 sec and hypochlorite sodium solution (NaOCl) at 2% for 2.5 min, and twice 
washed with sterilized distilled water in order to remove hypochlorite excess (Petrini 
1996; modified technique). Six surface sterilized discs were transferred in triplicate to 
each Petri dish containing Potato Dextrose Agar (PDA) + chloramphenicol (50 mg.L") 
to prevent bacterial growth. The plates were incubated at room temperature (28° + 2°C) 
and observed daily during 15 days for colony development. For asepsis control, 50 uL of 
water used to remove hypochlorite was plated in PDA to confirm surface disinfection 
(Pereira et al. 1993). Species identification was based on macro- and micro-structural 
characteristics of the colony according to Subramanian (1956) and Ellis (1971). One 
isolated P. crotalariae, lyophilized and conserved in mineral oil, is maintained in the 
Culture Collection Depot at Universidade Federal de Pernambuco (URM). 


Results 
Five isolates of P. crotalariae were obtained from the dry season collection 
(February 2010). 


Phaeotrichoconis crotalariae (M.A. Salam & P.N. Rao) Subram. Proc. 
Indian Acad. Sci., B 44: 2 (1956) PLATE 1 
Colony on malt extract agar, incubated for eight days at room temperature 
(28 + 2°C), showed well-developed, flocculate aerial mycelium, at first 
light orange, turning gray upon reaching maturity, reddish brown reverse. 
Conidiophores macronematous, mononematous, unbranched, straight or 
flexuous, brown, smooth, 19.2-141.6 x 3.6-4,8 um. Polytretic conidiogenous 
cells, terminal, integrated, merged, sympodial, cylindrical, with dark scars. 
Conidia enteroblastic, acrogenous, solitary, dry, fusiform to obclavate, 
elongated, rostrate, usually with 5-6 transverse septa, thick walled, smooth, 
with a dark brown scar at the base, 45-79.2 x 12-17.5 um. Appendix long, 
hyaline to pale brown, narrow (2.4 um diam.), 35-220.8 um long, 0-1 thin 
septum. Brown-dark sclerotia present. 
SPECIMEN EXAMINED: BRAZIL. PERNAMBUCO: SAO VICENTE FERRER, in healthy mature 
leaves of Vitis labrusca L. cv. Isabel, 11 Feb 2010, Lima (URM 6360 [culture conserved 
in mineral oil and lyophilized]). 
Notes: This is the second record of Phaeotrichoconis crotalariae from Brazil, 
and the first time it has been described on artificial culture medium. The fungus 
has not previously been recorded from Vitis. 


Acknowledgments 
The authors thank Francisco C.O. Freire (Embrapa Agroindustria Tropical, 
Brazil),and Edna D.M. Newman Luz (Ceplac/Cepec/Sefit, Brazil) for critical review. 


294 ... Lima, Bezerra & Cavalcanti 


Literature cited 

Chase AR. 1982. Dematiaceous leaf spots of Chrysalidocarpus lutescens and other palms in Florida. 
Plant Dis. 66: 697-699. http://dx.doi.org/10.1094/PD-66-697 

Ellis MB. 1971. Dematiaceous hyphomycetes. Commonwealth Mycological Institute, Kew, England. 
608p. 

Freire FCO, Bezerra JL. 2001. Foliar endophytic fungi of Ceara State (Brazil): a preliminary study. 
Summa Phytopathol. 27(3): 304-308. 

Miller JW. 1997, Plant pathology. Tri-ology Tech Report 36: 11-12. 

Mishra B, Prakash O, Misra AP. 1972. Incidence of Phaeotrichoconis crotalariae on Cyperus iria 
Linn. from India. Science and Culture 38: 371-372. 

Mohanan C, Sharma JK. 1988. Diseases of exotic acacias in India. J Trop Forestry 4: 357-361. 

Old KM, Harwood CE, Robson KJ, Haines MW, Solomon DJ. 1996. Foliar pathogens of tropical 
acacias in Australia. 11-19, in: KSS Nair et al. (eds). Impact of Diseases and Pests in Tropical 
Forests. Proceedings of IUFRO Symposium, Pecchia, Kerala, India. 

Pereira JO, Azevedo JL, Petrini O. 1993. Endophytic fungi of Stylosanthes. Mycologia 85: 362-364. 
http://dx.doi.org/10.2307/3760696 

Petrini O. 1996. Ecological and physiological aspects of host-specificity in endophytic fungi. 
87-100, in: SC Redlin, LM Carris (eds). Endophytic fungi in grasses and woody plants. St Paul: 
American Phytopathological Society Press. 

Ramakrishnan CK, Menon MR, Devi LR. 1972. Alternanthera sessilis R.Br. A new host of 
Phaeotrichoconis crotalariae (Salam and Rao) Subramanian comb.nov. Curr. Sci. 41: 751-752. 

Salam MA, Rao PN. 1954. A new species of Trichoconis on L. from Hyderabad-Dn. J Indian Bot 
Soc 33: 189-191. 


Subramanian CV. 1956. Phaeotrichoconis, a new genus of the Dematiaceae. Proc. Indian Acad. Sci., 
B 44:1-2. 


ISSN (print) 0093-4666 © 2012. Mycotaxon, Ltd. ISSN (online) 2154-8889 


MY COTAXON 


http://dx.doi.org/10.5248/120.295 
Volume 120, pp. 295-300 April-June 2012 


Studies on Wrightoporia from China 1. 
A new species from Hunan Province, South China 


JIA-JIA CHEN* & Hal-You Yu 


Institute of Microbiology, P.O. Box 61, Beijing Forestry University, Beijing 100083, China 
*CORRESPONDENCE TO: freejoly@yahoo.com.cn 


AxBstRAcTt — Wrightoporia nigrolimitata sp. nov. is described from Hunan Province, 
southern China. It is characterized by an annual growth habit, small pileate basidiocarps, a 
brown to black line between the tubes and context, a monomitic hyphal system with clamp 
connections, nondextrinoid hyphae, and finely asperulate amyloid basidiospores. A key to 
accepted Chinese Wrightoporia species is supplied. 


Key worps — Bondarzewiaceae, polypore, taxonomy, wood-inhabiting fungi 


Introduction 

Pouzar (1966) established Wrightoporia Pouzar for W. lenta (Overh. & 
J. Lowe) Pouzar, the type species. This genus is characterized by resupinate to 
pileate basidiocarps, annual to perennial growth habit, monomitic to trimitic 
hyphal system, and amyloid asperulate basidiospores (Pouzar 1966, Ryvarden 
1982). Of the 39 species described in or transferred to the genus worldwide 
(Buchanan & Ryvarden 2000; David & Rajchenberg 1987; Hattori 2003, 2008; 
Johansen & Ryvarden 1979; Lindblad & Ryvarden 1999; Loguercio-Leite et al. 
1998; Nunez & Ryvarden 2001; Rajchenberg & David 1990; Ryvarden 1975, 
1987, 2000), 14 species have been found in China (Cui & Dai 2006; Dai 2012; 
Dai & Cui 2006; Dai et al. 2011). 

Many new polypores have been recently described due to research on the 
diversity of wood-inhabiting fungi in southern China (Cui & Dai 2008, 2011; 
Cui et al. 2009, 2010, 201 1abc; Dai et al. 2010, 2011; Du & Cui 2009; Jia & Cui 
2011; Li & Cui 2010). During our study of wood-rotting fungi from Mangshan 
Forest Park, Hunan Province, we found one Wrightoporia species with a 
monomitic hyphal structure that cannot be identified to any known species. 
We describe it as a new species in this paper and provide an identification key 
to the 15 Wrightoporia species recorded in China. 


296 ... Chen & Yu 


Materials & methods 

Sections were studied microscopically according to Dai (2010) at magnifications 
< 1000x using a Nikon Eclipse E 80i microscope with phase contrast illumination. 
Drawings were made with the aid of a drawing tube. To present spore size variation, 
the 5% of measurements excluded from each end of the range are given in parentheses. 
Basidiospore spine lengths are not included in the measurements. Abbreviations include 
IKI = Melzer’s reagent, IKI- = negative in Melzer’s reagent, KOH = 5% potassium 
hydroxide, CB = Cotton Blue, CB- = acyanophilous, L = mean spore length (arithmetic 
average of all spores), W = mean spore width (arithmetic average of all spores), 
Q = variation in the L/W ratios between the specimens studied, and n = number of 
spores measured from given number of specimens. Special colour terms follow Petersen 
(1996). The studied specimens were deposited in herbaria as cited below. 


Taxonomy 


Wrightoporia nigrolimitata Jia J. Chen, sp. nov. FIG. 1 
MycoBank MB 564036 


Differs from other Wrightoporia species by its small pileate basidiocarps, presence of 
a black layer between tubes and context, a monomitic hyphal system, and generative 
hyphae that are nondextrinoid and dissolve in KOH. 


Type — China. Hunan Province, Yizhang County, Mangshan Forest Park, on angiosperm 

stump, 25.VI.2007, Li 1697 (holotype, BJFC; isotype, IFP). 

EryMoLocy — nigrolimitata (Lat.): referring to the black line separating the tubes and 

context. 
FruiTBopy — Basidiocarps annual, pileate, hard corky upon drying, without 
odor or taste; pileus semi-circular to irregularly formed, projecting up to 1 cm 
long, 2 cm wide, 4 mm thick at base. Pileal surface straw-yellow to honey- 
yellow at base, buff-yellow towards the margin, azonate, slightly sulcate or not; 
margin obtuse. Pore surface cream-buff to pinkish buff when dry; pores round 
to angular, 4-7 per mm; dissepiments thin to slightly thick, entire. Context buff 
to cinnamon-buff, hard corky, up to 1 mm thick. Tubes concolourous with pore 
surface, fibrous-tough, up to 3 mm long; a brown to black line present between 
tubes and context. 

HyPHAL STRUCTURE — Hyphal system monomitic; generative hyphae 
bearing clamp connections, IKI- or weakly dextrinoid in the black layer, CB-, 
hyphae in trama and context dissolved in KOH; hyphae in the black layer 
become reddish to dark brown in KOH. 

CONTEXT — Generative hyphae hyaline, thin- to slightly thick-walled, 
frequently branched, regularly arranged, 1.5-8 um in diam. 

TuBEs — Generative hyphae hyaline, thin- to slightly thick-walled, 
frequently branched, subparallel to parallel along the tubes, 2-7 um in diam; 
cystidia absent, fusoid cystidioles present, hyaline, thin-walled, 13-19 x 3.5-5 
um; basidia clavate, bearing four sterigmata and a basal clamp connection, 


Wrightoporia nigrolimitata sp. nov. (China) ... 297 


CD 
(> 
a, 
SS 


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|» rx. E> © 
Oo © 
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ee ee 
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Fic. 1. Microscopic structure of Wrightoporia nigrolimitata (drawn from the holotype). 
a: Basidiospores. b: Basidia and basidioles. c: Cystidioles. 
d: Hyphae from trama. e: Hyphae from context. 


13-25 x 4-6 um; basidioles in shape similar to basidia, but slightly smaller. 

Spores — Basidiospores ellipsoid, hyaline, thin-walled, mostly bearing one 
or two small guttules, finely asperulate, amyloid, CB-, 3-4 x 2-2.7(-2.9) um, 
L = 3.43 um, W = 2.42 um, Q = 1.42 (n = 30/1). 


REMARKS — Although most Wrightoporia species possess a dimitic hyphal 
system, only three others have a monomitic hyphal system: W. flava (Ryvarden) 


298 ... Chen & Yu 


A. David & Rajchenb., W. novae-zelandiae Rajchenb. & A. David, and 
W. porilacerata Log.-Leite et al. (David & Rajchenberg 1987, Loguercio-Leite et 
al. 1998, Rajchenberg & David 1990, Ryvarden 1975). 

Wrightoporia porilacerata also possesses pileate basidiocarps and similar 
sized (3-3.5 x 2-2.5 um) basidiospores but is distinguished from W. nigrolimitata 
by distinctly larger basidiocarps (< 13.5 x 8.7 x 2.6 cm), larger pores (1-3 per 
mm), no black line between tubes and context, and interwoven tramal hyphae 
(Loguercio-Leite et al. 1998). 

Like W. nigrolimitata, W. flava has pileate basidiocarps, nondextrinoid 
generative hyphae, and thin-walled asperulate amyloid basidiospores. However, 
W. flava is distinguished by possessing gloeoplerous hyphae, interwoven tramal 
hyphae, and lack of a black layer (David & Rajchenberg 1987, Ryvarden 1975). 

Except for a monomitic hyphal system, W. novae-zelandiae is unique in 
Wrightoporia by combining a gossypine brittle resupinate basidiocarp with 
gloeoplerous hyphae (Rajchenberg & David 1990). 

OTHER SPECIMENS EXAMINED — Wrightoporia flava. GABON. OGoovE-IvINDo 
PROVINCE, Makokou, Ipassa, XII.1974, LY 1733 (LY). TANZANIA. TANGA PROVINCE, 
Usambara Mts. Amani, Tanga distr., 18.11.1973, Ryvarden 10594 (O). MALAWI. 
SOUTHERN PROVINCE, Makwawa, 16.XI]I.1981, B.M. 342 (O). 

— W. novae-zelandiae. NEW ZEALAND. AUCKLAND, Waitakere Ranges, Kitekite 
Track, on Leptospermum sp., 19.IV.1989, P.K. Buchanan 89/114 (paratype, PDD 55206). 


— W. porilacerata. BRAZIL. PARANA StTaTE, Parangua, Guaraguacu, fazenda 
Sambaqui, on decayed dicotyledon trunk, 22.V1.1993, A.A.R. De Meijer 2805 (O). 


Key to species of Wrightoporia in China 


PRE phial Syston tn GO CRMC pa, scieg eis cheg ph feken hh Seba ti foe db Med hes W. nigrolimitata 
Litiyphalsystemndimiitic- OrgQcunittie eo. ee a hon Me kee hat Nee bie dhe Rar een banged 2 
2 Generative hyphae-withoutclainp:conmections 2%: 1108-1 ii.0%2 bites ites YE 3 
2, Generative hyphae with clamp connections. 0.85 vo kes hones abe eee Gaee as 5 
3. Basidiocarps pileate; basidiospores >7 um long... . W. radicata G.Y. Zheng & Z.S. Bi 
3. Basidiocarps resupinate to effused-reflexed; basidiospores <7 um long............ 4 
4. Basidiocarps without rhizomorphs .......... W. casuarinicola Y.C. Dai & B.K. Cui 
4; Basidiocarps with rhizOmorpns. 44.4 ja.. 5.6.5 esas esse beets eS W. rubella Y.C. Dai 
5: Basidiospores: > 5:9: (unr longa. 4. oe ia ee enone nee eg sleet S W. lenta 
DoDASIMOSPOLES, So 94 [ FIA TONS creek onthe cael h gaia dn cued geal) g seca erates erates eee 6 
Go SKe letal iyo Ae Geer UNGIG La pct iene ty aes ca, ean Oe ie cal Bea aly a Penna tama he natn 7 
Gs Skeletal hyplaedexeinG ide eee en en eee ee ee ee Ok ete d tie oe dee ec lel 8 
7. Cystidia and gloeocystidia absent....................006. W. aurantipora T. Hatt. 
Pakystidiatand, gloeoeystidia presents. sact:"sceuctss Sewn Bs Seewe Baes W. austrosinensis Y.C. Dai 
SiGloeoplerous-hyp lide pEeSent «shee vtcrmew, Wieew Mehr Piedra Web den eRe tern neterdern ef ion ber te 9 
SGlOsGplerausihyplige ADSeMt ss. yee res re sania aight armen a orien dated nw the CaN ease 10 


Wrightoporia nigrolimitata sp. nov. (China) ... 299 


9. Basidiocarps membranous; tramal skeletal hyphae 1-2 um in 


PAINS Ws. Os Sete No a Go RN Nas bes Nb ebay A bo W. avellanea (Bres.) Pouzar 
9. Basidiocarps tough; tramal skeletal hyphae 2-2.5 um in diam .. W. borealis Y.C. Dai 
10. Basidiospores >3.2 um wide ................ W. unguliformis Y.C. Dai & B.K. Cui 
LOU BAsiGsOsPOles. <32 NP WIeL «5 se tlenk wetlina tytn satire seth Nasa ten aside oe nmabireet waine 11 
Ph Skeletalthyphae ioteneruste dr 5 fi oy odie ng Lid aeek dinner d ald peek ai penen 42 behets tupetet 12 
IL Skeletal Ny phaerenceusted sts ft Me Adri. tRGot UeMte! LeRG wtamlor LEM URN id 13 
12. Basidiocarps annual; tramal skeletal hyphae 2-4 um in 

UAT ra Stas 5 iia lc ee es tae pete gies Rg ws sete al W. africana I. Johans. & Ryvarden 
12. Basidiocarps perennial; tramal skeletal hyphae 4-8 um in 

CHATS, corde ott scien coe Seed Vas yo ane Ya W. tropicalis (Cooke) Ryvarden 
13. Skeletal hyphae in the trama inflated, up to 6-9 um . W. luteola B.K Cui & Y.C. Dai 
13. Skeletal hyphae in the trama not inflated, up to5um............ 0c. eee eee 14 


14. Generative hyphae with both clamp connections and simple septa; cystidioles 
ADSOMES we leet iapten h.So tan talaga the atta tee W. gillesii A. David & Rajchenb. 
14. Generative hyphae only with clamp connections; fusoid cystidioles present 
thy Wheat, Btbetln Hekate, Hevaty ween WEN eee 8 W. japonica Nufiez & Ryvarden 


Acknowledgements 

We express our gratitude to Drs. Hai-Sheng Yuan (IFP, China) and Xiao-Yong 
Liu (HMAS, China) who reviewed the manuscript. The research was financed by the 
National Natural Science Foundation of China (Project No. 31170018) and Program for 
New Century Excellent Talents in University. 


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Cui BK, Dai YC, Yuan HS. 2010. Two new species of Phylloporia (Basidiomycota, Hymenochaetaceae) 
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ISSN (print) 0093-4666 © 2012. Mycotaxon, Ltd. ISSN (online) 2154-8889 


MYCOTAXON 


http://dx.doi.org/10.5248/120.301 
Volume 120, pp. 301-307 April-June 2012 


A new species of Bipolaris from Iran 


ABDOLLAH AHMADPOUR', ZEINAB HEIDARIAN', MARYAM DONYADOOST- 
CHELAN’, MOHAMMAD JAVAN-NIKKHAH’ & TAKAO TSUKIBOSHI’ 


"Department of Plant Protection, Faculty of Agricultural Sciences & Engineering, 
University College of Agriculture and Natural Resources, 
University of Tehran, Karaj, Iran 

? National Institute of Livestock and Grassland Science, 
Senbonmatsu 768, Nasushiobara, Tochigi 329-2793, Japan 

* CORRESPONDENCE TO: ahmadpour.abdollah@gmail.com 


ABSTRACT— Bipolaris salkadehensis sp. nov., isolated from the leaves of Sparganium 
erectum (Thyphaceae) and Cladium mariscus (Cyperaceae) collected in Iran, is described 
and illustrated. The new species is characterized by straight to slightly curved subcylindrical 
to fusoid conidia with end cells demarcated by thick dark septa and with monopolar and 
bipolar germination. Morphological data and ITS+5.8SrDNA sequence analyses support 
B. salkadehensis as a distinct species of Bipolaris. 


Key worps — morphology, taxonomy, fungus, Cochliobolus, phylogeny 


Introduction 

Bipolaris species are known to infect many kinds of grasses causing spots or 
flecks on leaves or stems and sometimes cause diseases on herbaceous plants 
such as Cactaceae and Musaceae (Ellis 1971, Sivanesan 1987). Since Sivanesan 
(1987) monographed the graminicolous species of the genus, several new 
Bipolaris species have been described (Chiang et al. 1989, Peng & Lu 1989, 
Sisterna 1989, Alcorn 1990, Sivanesan 1992, Alcorn 1996, Chen et al. 2000, Zhu 
et al. 2000, Deng & Zhang 2002, Jiang & Zhang 2008, Zhang & Li 2009). Recent 
molecular phylogenetic analyses have shown that Bipolaris and Curvularia are 
closely related to each other and comprise a different clade from Exserohilum 
(Berbee et al. 1999, Olivier et al. 2000). The rDNA-ITS sequences of (particularly 
the internally transcribed spacers ITS1 and ITS2) are useful tools for resolving 
taxonomic relationships within Bipolaris. 

In this study, we describe a new species of Bipolaris, which we compare both 
morphologically and phylogenetically with other species. 


302 ... Anmadpour & al 


Materials & methods 


Collection and isolation of the fungus 

Plant material was obtained from the Salkadeh village, Khoy city, West Azerbaijan 
province, in northwestern Iran in the summer of 2010. Leaf samples of Sparganium 
erectum L. (Thyphaceae) and Cladium mariscus (L.) Pohl (Cyperaceae) with brown oval 
to elliptical lesions with approximately 2-3 mm in length were collected and stored dry 
in a refrigerator at 5°C until use. A single spore produced on the surface-sterilized leaf 
under Nuv light on 12 h diurnal cycle at room temperatures 23 + 2°C (Sivanesan 1987) 
was transferred to tap water agar with autoclaved wheat straw (TWA+wheat straw) plates. 
Five isolates were obtained: Bi-1-3 from S. erectum, and Bi-4—5 from C. mariscus. For 
morphological inspection each isolate was transferred to Petri dishes containing PDA 
using a thin glass needle and incubated at 23 + 2°C under darkness; species descriptions 
are based on 10-14 day old cultures. Measurement and microphotographs were taken 
from slide mounts in lactophenol and lactophenol cotton blue using an Olympus light 
microscope (model BH2). The fungal isolates were compared with the descriptions 
of Ellis (1971, 1976), Sivanesan (1987) and reported species after 1987. Dried culture 
vouchers have been deposited in the herbarium in the Department of Plant Protection, 
Faculty of Agricultural Sciences & Engineering, University College of Agriculture and 
Natural Resources, University of Tehran, Karaj, Iran (TUPP). 


Molecular phylogenetic analyses 

Whole genomic DNA was extracted from the mycelium of each isolate grown on 
V8 juice agar by homogenization in a standard sodium dodecyl sulfate (SDS) detergent 
lysis buffer followed by a phenol : chloroform extraction and precipitation in ethanol 
with sodium acetate (Sambrook et al. 1989). The ITS1+5.8S+ITS2 rDNA region was 
amplified using the polymerase chain reaction (PCR) conditions and the ITS1 and 
ITS4 primer pair (White et al. 1990). Purified PCR products were sequenced by ABI 
PRISM 3100 automated sequencers (Applied Biosystems, Foster City, CA, USA). For 
phylogenic comparison, the GenBank sequences of 26 Bipolaris species, 5 Curvularia 
species, 2 Exserohilum species, 2 Drechslera species, and Alternaria alternata (Fr.) Keissl. 
(as outgroup) were included (Berbee et al. 1999). The DNA sequences were aligned using 
Clustal X version 1.8 (Thompson et al. 1997). Further visual alignments were done in 
Sequence Alignment (Se-Al) Editor version 2.0 (Rambaut 2000). The data were analysed 
phylogenetically using distance methods. The distance matrix was calculated using 
Kimura's two parameter method (Kimura 1980) and analyzed with the neighbor-joining 
(NJ) method (Saitou & Nei 1987) using the program PAUP* 4.0 beta 10 (Swofford 2002). 
Bootstrap values were generated with 1000 replicate heuristic searches to estimate 
support for clade stability of the consensus tree using the same program. Sequences 
have been deposited in the DNA Data Bank of Japan (DDBJ). 


Results 


New species 


Bipolaris salkadehensis Ahmadpour & Heidarian, sp. nov. Fic. 1 
MycoBaAnk MB 564565 


Differs from Bipolaris cynodontis in conidia with both monopolar and _ bipolar 
germination and conidial end cells that are demarcated by thick dark septa. 


Bipolaris salkadehensis sp. nov. (Iran) ... 303 


Fic. 1. Bipolaris salkadehensis. A, B. Conidiophores. C-G. Conidia. H-J. Germinating conidia. 
Scale bars = 10 um. 


Type: Iran. West Azerbaijan, Khoy City, Salkadeh village, on infected leaves of Sparganium 
erectum, 20 Sep 2010, coll. A. Ahmadpour Bi-1 (Holotype, TUPP1366 [dried culture]; 
DDBJ sequence, AB675490). 


ETyMOLoGy: referring to the type locality. 


Colonies in “[WA+wheat straw’ agar brown, velvety, floccose. Hyphae brown, 
septate, branched, smooth, 5-7 um wide. Stromata not formed. Conidiophores 
single or fasciculate in small groups, simple, pale brown to brown, cicatrized 
with scars often inflated and smooth, multiseptate, 225-590 um long, swollen 
to 7.5-15 um diam at base, then narrowing to 5-8.5 um diam (middle) and 
5-7.5 um diam (apex). Conidia pale brown to dark brown, subcylindrical to 
fusoid, occasionally obclavate to clavate, straight to slightly curved, smooth, 
end cells rounded, very pale and demarcated by thick, dark septa, (4—)6-8 
(-10) distoseptate, (32—)53-80(-93) x 12-15 um, hilum <2 um diam, sometimes 
slightly protruding. Germination of conidia is monopolar and bipolar. The 
primary septum in developing conidia is submedian, the second delimiting the 
basal cell, and the third distal. 


304 ... Anmadpour & al 


Cultural characteristics: Colony velvety, floccose, olivaceous-brown on 

PDA. 

ADDITIONAL MATERIAL EXAMINED: IRAN. WEST AZERBAIJAN, Khoy City, Salkadeh 

village, on infected leaves of Cladium mariscus, 20 Sep 2010, coll. A. Ahmadpour Bi-4 

(TUPP1362; DDBJ sequence, AB675491) and Bi-5, on infected leaves of Sparganium 

erectum, Bi-2-3. 
ComMENTs: Its conidial morphology clearly establishes our specimen as 
belonging to Bipolaris. It is morphologically similar to B. cynodontis, B. setariae 
(Sivanesan 1987), B. sesuvii (Zhang & Li 2009), and B. fusca (Jiang & Zhang 
2008). Bipolaris cynodontis is distinguished from the new species by its smaller 
conidia, absence of thick dark septa demarcating the end cells demarcated by 
thick dark septa, and with only bipolar germination (Sivanesan 1987). The 
conidia of B. setariae are somewhat similar — fusoid to navicular, pale to mid 
golden brown, 5-10 distoseptate, (45-)50-70(-100) x 10-15 um (Sivanesan 
1987) — but with end cells demarcated by unthickened hyaline septa and paler 
than those of B. salkadehensis. The conidia of B. sesuvii are olivaceous-brown 
to brown, basal cell concolorous or slightly pale, subcylindrical to broadly 
fusoid and rounded at ends, straight, smooth, 5-9 distoseptate, 52-77 x 
13-16 um (Zhang & Li 2009) and are narrower and have end cells demarcated 
by unthickened hyaline septa. The conidia of B. fusca, which are shorter and 
wider than those of B. salkadehensis, are straight or occasionally slightly 
curved, smooth, cylindrical or ellipsoidal, (3—)5-7(-11) distoseptate, 31-67 x 
11-20 um, yellowish brown to dark brown, with end cells often very pale and 
demarcated by thick dark septa (Jiang & Zhang 2008). 


Phylogenetic analysis & discussion 

PCR products of ITS+5.8S rDNA sequences were 506bp for isolate Bi-1 
and 505bp for isolates Bi-2-5. Since the Bi-2-5 sequences matched completely, 
the Bi-1 sequence was registered as DDBJ AB675490 and the Bi-4 as DDBJ 
AB675491. According to the neighbor-joining tree derived from ITS+5.8S 
rDNA sequences made in this study, the B. salkadehensis isolates formed 
a monophyletic group with a 95% bootstrap value close to B. cynodontis 
(AF163093) (Fic. 2). However, the 98.2% (497/506 bp) sequence similarity 
between B. salkadehensis and B. cynodontis indicates that those two species 
clearly differ. Moreover, despite similar conidial morphologies, B. salkadehensis 
formed a different group from B. setariae and B. sesuvii (Fic. 2). 

Berbee et al. (1999) used the ITS+5.8S rDNA sequences and a portion of the 
glyceraldehyde-3-phosphate dehydrogenase sequences to evaluate Cochliobolus 
(anamorphs Bipolaris) and proposed that Bipolaris be divided further, separating 
species with large, canoe-shaped, gently curving conidia (Cochliobolus Group 
1) from those with short, either straight or curved conidia lacking a gentle 
curve along the whole spore length (Cochliobolus Group 2), which intermix 


Bipolaris salkadehensis sp. nov. (Iran) ... 305 


Gipolaris zeicola AF158110 
Bipolaris victoriae AF 158109 
Bipolaris urochloae AF071334 
Gipolaris heveae AB179835 
Bipolaris peregianensis AF 158111 


76 


Bipolans oryzae X18122 
95, Bipolaris salkadehensis Bi-1 AB675490 
Bipolaris salkadehensis Bi-4 AB675491 
Gipolaris cynodontis AF163093 
Gipolaris stenospila AB119837 
Bipolaris luttrelhi AFO71350 Cochliobolus 
Bipolaris melinidis AF071350 Group 1 
Bipolaris zeae AF081452 
Bipolaris sorokiniana AF071329 
Gipolans sorghicola AF071332 
Bipolans setariae EF452444 
Bipolaris setariae GU290228 
Bipolaris bicolor AF120260 
Bipolaris maydis AF158108 
Bipolaris sacchari AF071318 
82 Bipolanis australiensis AF081450 
88 Bipolans hawaiensis AFO71324 
Gipolaris perotidis AF071320 
Cunulana gladioli AF071337 
Bipolaris ravenelii AF071321 
Cumulania lunata AF071339 
2 Cunularia gudauskasii AF071337 es px ti i : ¢ ae 
Cunlana afinis AF071335 
400 98| Sipolaris indica AF081449 
85 Bipolans sesuvi EF 175940 
Sipolanis portulacae AY004778 
98 Bipolaris kusanoi AF071352 
78 Bipolaris nodulosa GU073110 
Cumularia clavata AF071336 
400 Exserohilum minor AF071341 
Exserohilum rostratum AF071342 


This study 


100 


86 


99 Drechslera erythrospila AY004782 
Drechslera tritici-repentis AF071348 
Alternana alternata AF071346 


+1 0.02 substitutions/site 


Fic. 2. A neighbor-joining tree inferred from the ITS+5.8S rDNA sequences from 35 taxa. Bootstrap 
values for 1000 replicates are shown on the branches, and rDNA-ITS DDBJ accession numbers 
stand after the species names. The branch length is proportional to the number of base changes as 
indicated by the scale bar. Alternaria alternata (AF071346) is an outgroup. 


306 ... Anmadpour & al 


with Curvularia species. All B. salkadehensis isolates used in this study clustered 
in the Cochliobolus Group 1 subclade, which contains Bipolaris species large, 
canoe-shaped, gently curving conidia. Although the conidial morphology of 
B. salkadehensis is similar to B. cynodontis, B. setariae (Cochliobolus Group 1), 
and B. sesuvii (Cochliobolus Group 2), the phylogenetic analysis did not show 
a close relationship. The combined molecular-morphological analysis supports 
B. salkadehensis as an unreported new species. 

Although the teleomorph was not observed in cultures or on the natural 
host, the B. salkadehensis teleomorph relationships can be compared by using 
differences or similarity in conserved DNA sequences (e.g. ITS, Gpp, BRN1 
and mating type genes) with other known Bipolaris species to provide a more 
reliable classification system at the generic and species levels (Berbee et al. 
1999, Shimizu et al. 1998, Turgeon 1998). The MAT phylogenetic trees, along 
with the ITS and Gpp trees, can be used as a database from which to look back 
into evolutionary history in order to understand how different reproductive 
lifestyles arose (Turgeon 1998). 

Both morphological and phylogenetic evidence supports B. salkadehensis 
as a previously unreported species separate from other known Bipolaris. 
This is the first report of Bipolaris on the hosts S. erectum (Thyphaceae) and 
C. mariscus (Cyperaceae). 


Acknowledgments 

The authors wish to special thanks to Dr. Meng Zhang (College of Plant Protection, 
Henan Agriculture University, P. R. China) for kind guidance. We are greatly indebted 
to Dr. Guang-yu Sun (College of Plant Protection, Northwest A&F University, P. R. 
China) and Mr. Takuya Sakoda (Yokohama Plant Protection Station, Japan) for their 
critical reviewing of the manuscript. We also appreciate the corrections by Dr. Shaun 
Pennycook, Nomenclatural Editor, and suggestions by Dr. Lorelei L. Norvell, Editor-in- 
Chief. This work was financially supported by University of Tehran. 


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methods and applications. Academic, San Diego, California. 

Zhang JZ, Li MJ. 2009. A new species of Bipolaris from the halophyte Sesuvium portulacastrum in 
Guangdong Province, China. Mycotaxon 109: 289-300. http://dx.doi.org/10.5248/109.289 
Zhu MQ, Sun GY, Zhang TY. 2000. Studies on the genus Bipolaris of Guangxi province. Acta 

Agriculturae Boreali-occidentalis Sinica 9: 32-34. 
http://dx.doi.org/CN KI:SUN:XBNX.0.2000-03-008 


ISSN (print) 0093-4666 © 2012. Mycotaxon, Ltd. ISSN (online) 2154-8889 


MY COTAXON 


http://dx.doi.org/10.5248/120.309 
Volume 120, pp. 309-316 April-June 2012 


Notes on Xylophallus xylogenus (Phallaceae, Agaricomycetes) 
based on Brazilian specimens 


LARISSA TRIERVEILER-PEREIRA* & ROSA MARA BORGES DA SILVEIRA 


Depto. de Botanica, Universidade Federal do Rio Grande do Sul 
Av. Bento Goncalves 9500, Porto Alegre-RS, 91501-970, Brazil 


*CORRESPONDENCE TO: It_pereira@yahoo.com.br 


ABSTRACT — From morphological analysis of Phallus pygmaeus specimens collected in 
Brazil and reexamination of the holotype, we conclude that this species is a synonym of the 
previously described Xylophallus xylogenus and agree with the recently proposed synonymy. 
We describe new specimens of X. xylogenus from Northeastern Brazil and provide color 
photos and a key for the species of the small xylophilous members of Phallus, including 
Xylophallus. 


KEY worDs — gasteromycetes, herbarium revision, Mutinus, stinkhorns, tropical fungi 


Introduction 

Phallus pygmaeus was described from the tropical rainforest in Northeastern 
Brazil, growing on decaying wood (Baseia et al. 2003). The species is 
characterized by small basidiomata (< 15 mm high), a smooth receptacle with 
minute perforate apex, and lignicolous habitat. The remarkably small size of 
this phalloid is not exclusive to P pygmaeus, as other small species are known 
from the tropics, e.g., Phallus tenuis (E. Fisch.) Kuntze, P minusculus Kreisel 
& Calonge, and P. drewesii Desjardin & B.A. Perry (Calonge & Kreisel 2002, 
Calonge 2005, Desjardin & Perry 2009). 

Xylophallus xylogenus is a taxon with a problematic generic position. 
Originally described as Phallus xylogenus from Cayenne (French Guiana), 
its author (Montagne 1855) placed the species in sect. Mutinus, while some 
years later Schechtendal (1861) classified it in Phallus sect. Xylophallus Schltdl. 
Fischer (1898-99), who first proposed the combination Mutinus xylogenus, 
later erected the genus Xylophallus (Schltdl.) E. Fisch. for the species (Fischer 
1933a). Saenz et al. (1972) also contributed to the morphology and ontogeny of 
the species based on collections from Costa Rica. Since its original description, 
X. xylogenus has been ambiguously illustrated by different authors (Fic. 1). 


310 ... Trierveiler-Pereira & Silveira 


Fic. 1. Xylophallus xylogenus, redrawn from original publications: 
A-B. Montagne (1855), as Phallus xylogenus. C. Fischer (1933). 
D. Saenz et al. (1972). E. Baseia et al. (2003), as Phallus pygmaeus. 


Cheype (2010), who recently described and provided colour photos of 
X. xylogenus specimens from the type locality, proposed synonymizing 
P. pygmaeus with X. xylogenus. 

During field expeditions in the State of Pernambuco, Northeastern Brazil, 
specimens initially determined as P pygmaeus were collected in abundance 


Xylophallus xylogenus in Brazil... 311 


from rotten logs. Our analysis of this material and reexamination of the 
P. pygmaeus holotype leads us to confirm Cheype’s (2010) synonymy. 


Materials & methods 

Field expeditions were carried out in 2008 and 2009 in two reserves in the State 
of Pernambuco, Northeast Region of Brazil: Reserva Privada do Patriménio Natural 
(RPPN) Carnijé (Moreno; 8°10'00"S 35°05'15"W) and Parque Dois Irmaos (Recife; 
8°07'30"S 34°52'30"W). Both reserves, at altitudes at 20-180 m, are relict fragments 
of the Brazilian Atlantic rainforest. The sampled basidiomata were transported to the 
laboratory in plastic boxes (Lodge et al. 2004). Both fresh and dried basidiomata were 
examined. 

Macroscopic measurements and colors are based on fresh material. Colors are coded 
according to Kornerup & Wanscher (1978). Observations of microscopic characters 
were made under a light microscope on glass slides mounts (in 5% KOH) prepared from 
dried specimens. Voucher specimens are deposited in URM (Thiers 2011). 


Taxonomy 


Xylophallus xylogenus (Mont.) E. Fisch., Nat. Pflanzenfam., 2 Aufl., 7a: 96. 1933. 
FIGS 1-3 
= Phallus (Mutinus) xylogenus Mont., Ann. Sci. Nat., Bot., Sér. 4, 3: 137. 1855. 
= Mutinus xylogenus (Mont). E. Fisch., Nat. Pflanzenfam. 1(1**): 290. 1899. 

= Phallus pygmaeus Baseia, Mycotaxon 85: 78. 2003. 

BASIDIOMATA gregarious; when immature globose, subglobose to ovoid, 
sometimes tapering towards the base, glabrous, smooth or groovy, 2.5-3.5 mm 
high x 2.5-4 mm broad, yellowish brown (5D5, 5E5) at the apex, becoming 
lighter near to the base; at maturity, 6-14 mm _ high, including the volva. 
Votva yellowish brown, with irregular dehiscence, basal hyphal strand simple 
or multiple, whitish, penetrating the woody substrate or forming a network 
above it. PsEUDOSTIPE cylindrical, hollow inside, 5-7 mm high x 1.5-2.5 
mm broad, translucent white, indusium absent; externally ornamented by a 
reticulated structure composed of rectangular alveoles with prominent edges. 
RECEPTACLE conic-campanulate, surface smooth, adnate to the pseudostipe, 
with round or umbilicate apex, non or minutely perforate at maturity, 2.5-4 
mm high x 2.5-4 mm broad, concolor with pseudostipe; receptacle margin 
smooth to denticulate. GLEBA gelatinous, grayish green (1D5) to olive (1F7), 
very foetid. 

BASIDIOSPORES ellipsoid, smooth, thin-walled, hyaline or with greenish 
tints, 3-4 x 1.5 um. VoLtva composed of isodiametric to irregularly rectangular 
pseudoparenchymatous hyphae, hyaline, yellowish to brownish, slightly 
thick-walled, 7-15 x 6-13 um. PSEUDOSTIPE AND RECEPTACLE composed by 
isodiametric pseudoparenchymatous hyphae, hyaline, thin-walled or slightly 
thick-walled, 12-45 x 12-32 um. 


312 ... Trierveiler-Pereira & Silveira 


Y Ya tat 
Fic. 2. Xylophallus xylogenus in situ. 
A. Brazilian basidiomata (scale = 12 mm). B. Costa Rican basidiomata (photo by Clark L. Ovrebo). 


Xylophallus xylogenus in Brazil ... 313 


ECOLOGY & DISTRIBUTION: on rotten wood in French Guiana (Montagne 
1855, Cheype 2010), French Antilles (Guadeloupe; Cheype 2010), Suriname 
(Fischer 1933b), Costa Rica (Saenz et al. 1972, Saenz & Nassar 1982, Calonge et 
al. 2005), Brazil (as P pygmaeus; Baseia et al. 2003, Baseia et al. 2006, Leite et al. 
2007), Peru and Ecuador (Gémez & Gazis 2006). 

SPECIMENS EXAMINED: BRAZIL. PERNAMBUCO, GUuRJAU, 28.VI.2002, leg. Baseia & 
Gibertoni (URM 77078, holotype of Phallus pygmaeus); MORENO, RPPN Carnijé, 
17.V1.2008, leg. Trierveiler-Pereira 10, 11 (URM 80261, 80262); 08.VII.2008, Trierveiler- 
Pereira 83, 84 (URM 80264, 80265); 14.VIII.2008, Trierveiler-Pereira 186, 187 (URM 
80269, 80270); 23.1X.2008, Trierveiler-Pereira 148, 149 (URM 80267, 80268); 16.X.2008, 
Trierveiler-Pereira (URM 80271); 23.1.2009, Baltazar & Coimbra 206 (URM 80272); 
12.III.2009, Trierveiler-Pereira 207, 208 (URM 80273, 80274); 21.V.2009, Trierveiler- 
Pereira 226, 227 (URM 80275, 80276); ReciFE, Parque Dois Irmaos, 07.VII.2008, 
Trierveiler-Pereira 67 (URM 80263); 12.VIII.2008, Trierveiler-Pereira 142 (URM 
80266). 
REMARKS: Desjardin & Perry (2009) noted that P xylogenus was a long forgotten 
name. Various authors have illustrated it differently (Fic. 1). Montagne’s (1855) 
illustration (Fic. 1B), which is representative, depicts its xylophilous gregarious 
habit, while Montagne’s detailed figure of a single basidiome (Fic. 1A) is quite 
imaginary, showing the receptacle margin distant from the pseudostipe and the 
pseudostipe with a shallow reticulum. The illustration in Fischer (1933a), based 
on original material, is more realistic and the pseudostipe with deep alveoles is 
well represented (Fra. 1C). 

Lloyd’s (1907) photo of the original material shows that the upper basidiome 
of the type was not well preserved, leading him to describe the species as having 
a “capitate, globose mass of gleba”. Dennis (1970) repeated this information, 
probably based on Lloyd's description. Lloyd’s belief that Xylophallus was a 
synonym of Mutinus has been wrongly propagated through the literature 
during the 20th century. 

Saenz et al. (1972) accompany their good detailed description of X. xylogenus 
from Costa Rica with an illustration of the receptacle with an umbilicate apex 
(Fic. 1D) and perforated apex (approximately 100 um in diam). Baseia et al. 
(2003) distinctly illustrated the receptacle pore (Fic. 1E). Among the examined 
materials, we found specimens with rounded to umbilicate apices, minutely 
perforated or not. We believe that the perforated apex probably represents the 
mature receptacle. 

Calonge et al. (2005), basing their observations on Dring (1973), cited X. 
xylogenus as cosmopolitan. However, Dring noted distribution only for the 
entire genus Mutinus, not for this species in particular. According to Gomez 
& Gazis (2006) and elsewhere, although X. xylogenus may be common in 
neotropical forests, the species rarely collected due to its minute size and 
ephemeral nature. 


314 ... Trierveiler-Pereira & Silveira 


Fic. 3. Xylophallus xylogenus. 
A. Single basidiome (scale = 2.5 mm). B-D. Microscopical structures (scale = 10 um): 
B. Pseudoparenchymatic hyphae from the pseudostipe. 
C. Pseudoparenchymatic hyphae from the volva. D. Basidiospores. 


We agree with Saenz et al. (1972) in separating X. xylogenus from Mutinus, 
since the gleba develops on a receptacle externally modified from the 
pseudostipe. Although the xylophilous habit of X. xylogenus is observed in 
other Phallus species, we prefer to retain the species in Xylophallus until further 
molecular analyses are carried out. 


Xylophallus xylogenus in Brazil ... 315 


Key to small xylophilous phalloid species 


la. Receptacle smooth, minutely perforated; pseudostipe with rectangular alveoles 
PIN COLEO PICS) oF voter Muh Ae tere roe man uirh, Muel nied oth Xylophallus xylogenus 


1b. Receptacle reticulate, clearly perforated with a conspicuous pore; pseudostipe 
mintitely reticulate-Orspongy~ 264 2.500 2.n Goth Gn Pee teh 8S cle c8S ae tee ao 2 


2a. Pseudostipe yellow, basidiomata 70-100 mm high (China, Japan, Indonesia, 
Srp Wan kes, Aro pial FATE CA) 4 at actg dst oxteg ty isgeten dressed even Hest geen dyitgctt Phallus tenuis 


2b. Pseudostipe white, basidiomata smaller, < 45 mm high.....................0.. 3 


3a. Pseudostipe spongy, 25-33 mm high; basidiospores 2-3 um long (Tanzania, 
CASIET MATIC Ae A 8) F589 Ate RO RL Ee aa MOE oe P. minusculus 


3b. Pseudostipe reticulate-lacunose, 20-45 mm high; basidiospores 3-3.5(-3.8) um 
lotisstSao Pome Western AaPiCa)S 5 ct. Yecacecn iva Ma Hevea Nova che Bona dba yesua’s P. drewesii 


Acknowledgments 

We thank RPPN Carnijé and Parque Dois Irmaos (Pernambuco, Brazil) for permission 
to collect; Juliano M. Baltazar and Victor R.M. Coimbra for helping during field work; 
PPGBF-UFPE and Dra. Leonor Costa Maia, curator of Herbarium URM; CNPq and 
CAPES (Brazil) for providing fellowship to the first author. We express sincere gratitude 
to Clark L. Ovrebo (U.S.A.) for his photo and Gabriel Grilli (Argentina) for his assistance 
in scanning photo slide. We are grateful to Marc Stadler (Germany), Jean-Louis Cheype 
(France) and Juliano M. Baltazar (Brazil) for suggestions to improve the manuscript; 
Jacques Fournier (France) for sending useful literature. Dr. Eduardo R. Nouhra (IMBIV, 
Cordoba, Argentina), Maria Luciana H. Caffot (IMBIV, Cordoba, Argentina) and Taiga 
Kasuya (University of Tsukuba, Ibaraki, Japan) are acknowledged for critically reading 
the manuscript. 


Literature cited 

Baseia IG, Gibertoni TB, Maia LC. 2003. Phallus pygmaeus, a new minute species from a Brazilian 
tropical rainforest. Mycotaxon 85: 77-79. 

Baseia IG, Calonge FD, Maia LC. 2006. Notes on the Phallales in the Neotropics. Bol. Soc. Micol. 
Madrid 30: 87-93. 

Calonge FD. 2005. A tentative key to identify the species of Phallus. Bol. Soc. Micol. Madrid 29: 
9. 

Calonge FD, Kreisel H. 2002. Phallus minusculus sp. nova from tropical Africa. Feddes Repert. 
113(7-8): 600-602. 

Calonge FD, Mata M, Carranza J. 2005. Contribuicién al catalogo de los Gasteromycetes 
(Basidiomycotina, Fungi) de Costa Rica. Anales del Jardin Botanico de Madrid 62(1): 23-45. 

Cheype J-L. 2010. Phallaceae et Clathrus récoletés en Guyane francaise. Bulletin Mycologique et 
Botanique Dauphiné-Savoie 197: 51-66. 

Dennis RWG. 1970. Fungus flora of Venezuela and adjacent countries. Kew Bulletin Additional 
Series 3. Vaduz, J. Cramer. 

Desjardin DE, Perry BA. 2009. A new species of Phallus from Sao Tomé, Africa. Mycologia 101(4): 
545-547. http://dx.doi.org/10.3852/08-166 


316... Trierveiler-Pereira & Silveira 


Dring DM. 1973. Gasteromycetes, in Ainsworth GC, Sparrow FK, Sussman AS (eds.) The Fungi 
IVB. New York, Academic Press, pp. 451-478. 

Fischer E. 1898-99. Phallineae. 276-296, in: A Engler, K Prantl (eds). Die natirlichen 
Pflanzenfamilien, Teil 1(1**). 

Fischer E. 1933a. Phallineae. 76-108, in: A Engler, K Prantl (eds). Die natirlichen Pflanzenfamilien, 
2 Aufl., Vol. 7a. 

Fischer E. 1933b. Gastromyceteae Stahelianae. Ann. Mycol. 31(3): 113-125. 

Goémez LD, Gazis R. 2006. Dos Gasteromycetes (Basidiomycotina, Fungi) del Peru. Brenesia 65: 71. 

Kornerup A, Wanscher JH. 1978. Methuen handbook of colour. 3rd ed. London, Eyre Methuen. 

Leite AG, Silva BDB, Araujo RS, Baseia IG. 2007. Espécies raras de Phallales (Agaricomycetidae, 
Basidiomycetes) no Nordeste do Brasil. Acta Botanica Brasilica 21(1): 119-124. 
http://dx.doi.org/10.1590/S0102-33062007000100011 

Lloyd CG. 1907. Mycological Notes 26. Concerning the Phalloids. Mycological Writings 2: 
325-337. 

Lodge DJ, Ammirati JA, O’Dell TE, Mueller GM, Huhndorf SM, Wang CJ, Stokland JN, Schmit 
JP, Ryvarden L, Leacock PR, Mata M, Umania L, Wu Q, Czederpiltz DL. 2004. Terrestrial and 
lignicolous macrofungi. 127-172, in: GM Mueller et al. (eds). Biodiversity of fungi. Inventory 
and monitoring methods. Boston, Elsevier Academic Press. 

Montagne C. 1855. Cryptogamia Guayanensis. Ann. Sci. Nat., Bot., Sér. 4, 3: 91-144. 

Saenz JA, Nassar M. 1982. Hongos de Costa Rica: Familias Phallaceae y Clathraceae. Revista Biol. 
Trop. 30(1): 41-52. 

Saenz JA, Nassar M, Morales MI. 1972. Contribution to the study of Xylophallus xylogenus. 
Mycologia 64(3): 510-520. http://dx.doi.org/10.2307/3757868 

Schlechtendal DFL. 1861. Eine neue Phalloidee, nebst Bemerkungen uber die ganze Familie 
derselben. Linnaea 31: 101-194. 

Thiers B. 2011 [continuously updated]. Index Herbariorum: a global directory of public herbaria 
and associated staff. New York Botanical Garden's Virtual Herbarium. 
http://sweetgum.nybg.org/ih/ [accessed September 2011] 


ISSN (print) 0093-4666 © 2012. Mycotaxon, Ltd. ISSN (online) 2154-8889 


MYCOTAXON 


http://dx.doi.org/10.5248/120.317 
Volume 120, pp. 317-329 April-June 2012 


Plectania seaveri (Ascomycota, Pezizales), 
a new discomycete from Bermuda 


MATTEO CARBONE ?*, CARLO AGNELLO ” & SCOTT LAGRECA ° 


’ Via Don Luigi Sturzo 173, I-16148, Genova, Italy 

? Via Antonio Gramsci 11, I-72023, Mesagne (BR), Italy 

° Plant Pathology Herbarium, Dept. of Plant Pathology & Plant Microbe Biology, 
Cornell University, Ithaca, NY 14853, USA 


* CORRESPONDENCE TO: Matteocarb@hotmail.com 


ABSTRACT— Plectania seaveri sp. nov. is described after revision of Bermudian material 
identified by Seaver under the name Bulgaria melastoma. The only known host for this new 
species is the endemic Bermudian tree Juniperus bermudiana (Bermuda cedar), a species 
whose numbers have greatly reduced over the past 70 years due to the introduction of invasive 
scale insects. As this new fungus has not been collected since 1940, it may now be extinct. 
Color pictures of fresh and dried specimens, microscopic morphology, and a black and 
white drawing of hymenial elements are included. Related, excluded, and doubtful species of 
Plectania are also discussed. 


KEY wWoRDS— ascomycetes, Donadinia, taxonomy 


Introduction 

When the senior authors (MC, CA) began their revision of the genus 
Plectania Fuckel, they realized that the Bermudian collections reported by 
Seaver (1928, 1942) and Seaver & Waterston (1946) under the name Bulgaria 
melastoma [i.e., Plectania melastoma (Sowerby) Fuckel] required further study. 
After publication of Carbone et al. (2012) and Agnello & Carbone (2012), 
the need to re-examine these collections was even clearer. These early studies 
strongly indicated that Seaver’s Bermudian collections might represent a new 
taxon, and so, joined by the third author (SLG), we examined all Bermudian 
material of this taxon in the Plant Pathology Herbarium of Cornell University 
(CUR): 

Our revision rapidly confirmed that Bermudian material of Bulgaria 
melastoma in CUP does not belong to B. melastoma but in fact represents a 
strikingly different, undescribed species. We here name this species in honour 


318 ... Carbone, Agnello & LaGreca 


PiaTE 1. FJ. Seaver in his office at NY Botanical Garden (photographed 1930-35). Photo by 
H.M. Fitzpatrick, from the Cornell University Plant Pathology Herbarium photograph collection. 


of the original researcher for most of this material, the American mycologist 
Fred Jay Seaver (PLATE 1). 


Materials & methods 

Microscopic characters are based on dry specimens. Two optical microscopes were 
used: Olympus CX41 trinocular and Optika B353 trinocular with plan-achromatic 
objectives 4x, 10x, 40x, 60x, and 100x in oil immersion. Microscopic pictures were made 
using a Nikon 4500 camera and a Nikon Coolpix. Primary mounting media were Melzer’s 
reagent, cotton blue, and Congo red. Water mounts were used for all measurements 
and observations of pigments. At least 30 spores were measured from each examined 
apothecium. SEM observations were made using a Hitachi S-4000 scanning electron 
microscope (SEM) at the University of Florida Core EM facility (Gainesville, Florida, 
USA). A piece of the hymenium from the holotype was mounted on aluminum stubs 
with adhesive graphite tabs and sputter coated with a gold-palladium alloy for 60 s. 


Taxonomy 


Plectania seaveri M. Carbone, Agnello & LaGreca, sp. nov. PL. 2-6 
MycoBank MB 563704 


“Bulgaria melastoma” sensu Seaver, N. Amer. Cup-fung., 
Operc.: 198, 1928, Bermudian records. 


Plectania seaveri sp. nov. (Bermuda) ... 319 


Differs from Plectania melastoma in the absence of macroscopic orange-red warts, more 
stipitate apothecia, and ellipsoid and differently ornamented spores; from P. nannfeldtii 
and P helvelloides in a very different spore ornamentation; and from P lusitanica in 
larger spores and more pronounced ornamentation. 


Type: Bermuda, Walsingham, ca. 150 fruitbodies on bark of Juniperus bermudiana, 
20 January 1922, leg. H.H. Whetzel, Bermuda Fungi no. 188 [Holotype, CUP-Whetz. 
B.F.-0188, as Bulgaria melastoma]. 


Erymo.oey: In honor of the American mycologist, Fred Jay Seaver. 


MACROCHARACTERS (based on dried specimens) — APOTHECIA gregarious to 
subcespitose, mostly long stipitate, nearly globose and closed at first, expanding 
as they mature; base attached to substrate with very numerous dark black, 
threadlike filaments (subiculum); cup <18 mm across and <6 mm high; margin 
mostly entire: HyMENIUM blackish to black, apparently smooth; EXTERNAL 
SURFACE blackish to black, with a dark brownish to purplish-amaranth shade, 
rough, gently wrinkled; stipe <16 mm high and <2 mm thick, solid, black; the 
length varying depending on the point of growth from the substrate; FLESH 
leathery, darker near external surfaces. 

MICROCHARACTERS — ASCI 350-400 x 14-15 um, cylindrical, operculate, 
with a slightly excentric operculum, inamyloid, eight-spored, with walls <1 um 
thick and a tapered, flexuous base with a generative hypha that is connected 
more or less distantly from the basal septum; PARAPHYSES not, or only slightly, 
exceeding the length of the asci, 1.5-2(-2.5) um wide, cylindrical, septate, 
sometimes anastomosing, branched, pale brownish, multiguttulate by very small 
oil drops; the apex usually more or less diverticulate and lobed; an extracellular, 
brownish, amorphous pigment present in the upper part (observed in water 
mounts), uniting them in bundles; seraE (hymenial hairs) cylindrical, as long 
as the paraphyses, (3-)3.5-4.5 um wide, with a simple to slightly subcapitate 
apex and single septum at the very base, pale brownish; sporgs ellipsoid, 22-27 
x 10-12 um, most frequently 23-25 x 10-11 um, very few spores observed 
<31 um long, Q = (1.8-)2-2.7(-3), subhyaline to light yellowish, filled with 
a granular content and sometimes with few slightly bigger oil drops, walls <1 
um thick; spore sculpturing of small but well-defined round warts <0.8 um 
broad and 0.6 um high, visible also at low magnifications in optical microscopy; 
confirmed by the SEM, showing round isolated warts; very young spores are 
round, smooth and thick walled; suBHYMENIUM composed of a dense textura 
intricata of cylindrical, frequently septate, hyphae, with thickened, more or 
less dark brown walls; at low magnifications, it appears uniformly brownish to 
brown and not so sharply differentiated from the upper layer of the medullary 
excipulum; MEDULLARY EXCIPULUM of textura intricata with cylindrical, 
septate hyphae, 5-11 um wide, with walls on average 0.5-0.8 um thick; two 
main layers can be recognized: (i) an upper one with more brownish, denser, 
less gelified hyphae with a parallel orientation to the hymenial surface, (ii) a 


320 ... Carbone, Agnello & LaGreca 


PLaTE 2. Plectania seaveri. Upper row: CUP-033234: fresh specimens. Photo by FJ. Seaver: 
Bottom row: dried specimens of holotype collection. Photos by M. Carbone. Bar = 10 mm valid 
for bottom row only. 


middle-lower one with lighter to yellowish, more loosely interwoven hyphae 
immersed ina subgelatinous matrix; ECTAL EXCIPULUM of textura subglobulosa- 
angularis to textura angularis made up of elements <20 um wide and/or high, 
very dark brown due to the colored walls and the presence of an incrusting 


Plectania seaveri sp. nov. (Bermuda) ... 321 


brown pigment. In the very outer part the pigmentation becoming more 
amber-like and crystalline; EXTERNAL HAIRS mainly of two types, although 
intermediate forms occurring; (i) cylindrical, septate, hyaline, thin walled, 
heavily encrusted hyphoid hairs, <2.5 um wide; (ii) cylindrical, infrequently 
septate, <6 um wide, very long, mainly straight, smooth to very finely encrusted 
(only in the basal part) by the same kind of crystalline pigment, and brown 
due to an epimembranaceous pigmentation, with walls thickened <0.8 um; in 
non-squashed mounts, they are mainly lying on the external surface, and it is 
not easy to determine their entire length; susicuLUM made up of cylindrical, 
sometimes very slightly flexuous and/or notched, 6-9 um wide, <3 mm (or 
more) long, septate hyphae, brown due to an epimembranaceous pigmentation, 
with walls thickened <0.8 um, mainly smooth but in places a very light greenish 
extracellular pigment is present. 

ECOLOGY & DISTRIBUTION — gregarious to subcespitose, on bark of 
Bermuda cedar (Juniperus bermudiana L.). January and February. Known only 
from the Bermuda Archipelago. 

ADDITIONAL COLLECTIONS EXAMINED: Mature collections: BERMUDA, Agricultural 
Station, eleven fruitbodies on bark of Juniperus bermudiana on ground, 4 February 
1926, leg. HLH. Whetzel, Ogilvie & FJ. Seaver (CUP-033235); six fruitbodies on J. 
bermudiana, 15 January 1926, leg. H.H. Whetzel, Ogilvie & FJ. Seaver (CUP-034615). 
Burts Island, four fruitbodies on fallen tiny twigs of J. bermudiana, 9 February 1926, leg. 
H.H. Whetzel, Ogilvie & FJ. Seaver (CUP-033236). 

Immature collections: BERMUDA, Agricultural Station, on bark of Juniperus 
bermudiana on ground, 12 January 1926, leg. H.H. Whetzel, Ogilvie & FJ. Seaver (CUP- 
033231); on bark of living J. bermudiana, 28 January 1926, leg. H.H. Whetzel, Ogilvie & 


EJ. Seaver (CUP-033234). Harrington house, on fallen bark of J. bermudiana on ground, 
23 January 1926, leg. H.H. Whetzel, Ogilvie & FJ. Seaver (CUP-033233). 


Discussion 


PRELIMINARY NOTES — The first hint of the existence of these Bermudian 
collections can be found in Seaver’s note (1928) under Bulgaria melastoma: 
“What appears to be this species is very abundant in Bermuda on the bark 
of Bermuda cedar, Juniperus bermudiana, but never on anything else. Since 
the host is an endemic species, the fungus may also be found to differ and 
be itself endemic. The writer has not yet had the opportunity to clear up this 
point but reference will be made to it in later publications” In Seaver (1942), 
again under Bulgaria melastoma: “Range extended to Bermuda (FJ. Seaver & 
H.H. Whetzel)”. And lastly, in Seaver & Waterston (1946): > However one of us 
(Seaver 1928: 198, 1942: 320) has shown that the Bermuda species is referable 
to Bulgaria melastoma (Sow.) Seaver. Figure 1 (upper) shows this species on 
rotten bark of Juniperus bermudiana L., from roots of living trees exposed at 
soil level, Walsingham, Bermuda, Jan. 20, 1922, H.H. Whetzel Bermuda Fungi 
No. 188. This plant is characterized by apothecia which are frequently stipitate, 


322 ... Carbone, Agnello & LaGreca 


PLATE 3. Plectania seaveri (holotype). a—b: spores in lactic Cotton blue. c-e: spores in Congo 
red. f-h: spores in water mount. Bars: a, b, d, e, h = 10 um; c = 2,5 um; f, g = 5 um. Photos by 
M. Carbone. 


with almost smooth, hyaline, ellipsoidal spores, 20-25 x 9-10 uw”. In Waterston 
(1947) we find only a list of the collections studied here, under Bulgaria 
melastoma, and nothing is added from a taxonomic point of view. 

Korf (1957) seems to have been the first mycologist to realize that these 
collections might represent a new taxon: “...the species of “Bulgaria” described 
by Seaver and Waterston (1946: 182) from Bermuda, which appears to be 
undescribed...” While revising the cited material we found that Denison (Korf’s 
student) also noted Seaver’s misinterpretation, for in collection CUP-033235, 
there is a handwritten annotation noting, “This is not Bulgaria (Plectania) 
melastoma. Note, for example, the delicately sculptured spores, the larger 
apothecia and longer stipe. W C Denison 2 Nov 1961”. 

Manyyears before, Boedijn (1932) suggested that Seaver’s Bermudian Bulgaria 
melastoma (Seaver 1928) might be conspecific with Sarcosoma thwaitesii (Berk. 
& Broome) Petch [= Galiella thwaitesii (Berk. & Broome) Nannf.], mainly 


Plectania seaveri sp. nov. (Bermuda) ... 323 


because Petch (1910) recorded S. thwaitesii from Ceylon, growing on Juniperus 
bermudiana. Although Seaver & Waterston (1946) definitely misinterpreted 
Bulgaria melastoma, they demonstrated as well that Boedijn (1932) was wrong. 
Galiella thwaitesii is undoubtedly a very distinct and different taxon. For further 
details about Galiella thwaitesii see Le Gal (1953). 


SPECIES DELIMITATION — Plectania melastoma, in its original sense, is definitely 
a very different species from the material studied here. As demonstrated by 
Agnello & Carbone (2012) the macroscopic, vivid orange-red, external warts 
are always present, and still visible to the naked eye even after 150 years; in 
addition, the apothecia are at most substipitate and never definitely stipitate 
as in P. seaveri. Microscopically, there are more numerous and immediately 
appreciable differences. For example, in P. seaveri the spore sculpturing is 
delicate but, at the same time, very distinct and pronounced. It is easily visible 
in water mounts (1000x, oil immersion). In contrast, in P melastoma, the spores 
are far less sculptured and have very different warts that are barely visible, 
even in 1000x Cotton blue mounts. The spore shapes are also quite different 
— mostly subfusoid in P melastoma, ellipsoid in P. seaveri. Lastly, P melastoma 
produces only one kind of external hairs and a differently coloured crystalline 
pigmentation that is mostly a vivid orange-red-amber, not pale brownish to 
dark amber as in P seaveri. 

Unlike Plectania melastoma, P. seaveri surely belongs to Plectania sect. 
Donadinia (Bellem. & Mel.-Howell) M. Carbone & Agnello. In this section, 
Carbone et al. (2012) recognize only three stipitate species with more or less 
defined ornamented ellipsoid spores: P. helvelloides (Donadini et al.) Donadini, 
P. nannfeldtii Korf, and P. lusitanica (Torrend & Boud.) M. Carbone et al. As 
pointed out by Carbone et al. (2012), while all these species have extremely 
similar gross macroscopic morphologies, excipular anatomies, and external 
hairs, they can clearly be distinguished based on the combination of spore 
size and spore sculpturing. In fact, if on one hand we observe that spore 
measurements show a continuum from a minimum of 16 um (in Plectania 
lusitanica) to a maximum of 38 um (in Plectania nannfeldtii), on the other hand 
we are presently unaware of any populations that show a simultaneous overlap 
of spore size range and sculpturing. SEM and optical observations also support 
this morphological distinction. 

The well-known American “snowbank mushroom, Plectania nannfeldtii, can 
be immediately differentiated by its different ecology, geographical distribution, 
longer spores, and different episporium (e.g. Seaver 1928; Miller 1967; Smith et 
al. 1981; Arora 1986; Tylutki 1993; Li & Kimbrough 1995). Plectania lusitanica 
surely shares a similar ornamentation but differs in its smaller spores and a 
much less pronounced spore sculpturing that is barely visible in optical 
microscopy (data taken from many spore prints). Lastly, P helvelloides spores 


324 ... Carbone, Agnello & LaGreca 


PiateE 4. Plectania seaveri (holotype). a—-b: spores. c: pole of the spore. d: spore sculpturing. 
Bars a—b = 5 um; c-d = 1 um. SEM courtesy of Mark Whitten and the Electron Microscopy Core 
Facility at the University of Florida (Gainesville, USA). 


(22-28 x 9-13 um; Donadini et al. 1973, Carbone et al. 2012), which might be 
regarded as the closest to P. seaveri in size, have a different ornamentation type 
— relatively easy to see in cotton blue (1000x), but smaller, differently shaped, 
and not easily detectable in outline. 

The spore sculpturing seen in Plectania seaveri is extremely similar to that 
found in Galiella Nannf. & Korf, which, however, produces very different 
fleshy large gelatinous apothecia; for a quick survey, see Boedijn (1932, as 
Sarcosoma), Le Gal (1953, as Sarcosoma), Korf (1957), Cao et al. (1992), and 
Zhuang & Wang (1998). The same can be said for the genus Neournula Paden 
& Tylutki, previously placed in Sarcosomataceae Kobayasi but now transferred 
into a different family, Chorioactidaceae Pfister. Neournula does indeed possess 
similarly warted spores but is distinct from Plectania in many other features 
(see Pfister et al. 2008). 


Plectania seaveri sp. nov. (Bermuda) ... 325 


p eh 


see 


= 
oe 


Pate 5. Plectania seaveri (holotype). a: section of the apothecium; b: medullary excipulum in water 
mount; c: bundles of paraphyses; d: seta (hymenial hair); e: ascus tip; f: medullary excipulum in Congo 
red (magnified); g—h: hyphoid excipular hairs; i: excipular hairs; j: ectal excipulum; k: subiculum; 
1: encrusted cells of the margin; m: crystals of excipular pigment. Bars: a = 100 um; b = 20 um; 
c, e; f, j = 10 um; d, g, h, i, k, 1 = 5 um. Photos by M. Carbone. 


EXTRALIMITAL AND DOUBTFUL SPECIES — Mention must be made here of two 
other Plectania species described from the Americas. Plectania coelopus (Mont.) 
Sacc. was described from Chile and, according to the iconotype and original 
description, it surely belongs to sect. Donadinia. We agree with Le Gal (1958) 
and Cabello (1988) that P. coelopus must be regarded as a doubtful species until 


326 ... Carbone, Agnello & LaGreca 


PLaTE 6. Plectania seaveri (holotype). a: section of the cup; b: spores; c: ascus; d: paraphyses; 
e: seta (hymenial hair); f: excipular hairs. Bars: a = 100 um; b = 10 um; c-f = 10 um. Drawing by 
C. Agnello. 


its real identity is investigated and/or it is typified with topotypical (or other 
Chilean) material. 

Another doubtful American taxon is Plectania rimosa Peck, described from 
California (Peck 1903). Seaver (1942) treated it as a “doubtful species’, and due 
to the (apparent) lack of original material, he stated that this species might 
represent Paxina corium (O. Weberb.) Seaver [= Helvella corium (O. Weberb.) 
Massee]. As the protologue does not fit Helvella corium, we disagree and regard 
Plectania rimosa as a nomen dubium. 


Plectania seaveri sp. nov. (Bermuda) ... 327 


During our literature searches, we have found other unclear species 
seemingly in need of revision (e.g., Neournula helvelloides sensu Zhuang & 
Wang (1998) and Plectania nannfeldtii in the sense of the Chinese authors Teng 
(1963), Tai (1979), and Bi et al. (1990) and the Japanese author Otani (1973). 
Future studies will address the misapplications of these names. 


Eco.toecy — All known Plectania seaveri specimens were collected from rotting 
bark of Juniperus bermudiana, an endemic Bermudian tree that historically 
dominated the upland forests of the Bermuda Archipelago (Thomas 2004). Most 
collections of this fungus were made in the 1920s, with the last known specimen 
collected in 1940 (Waterston, 1947). A search of 125 fungus specimens from the 
last major Bermudian mycological expedition collected by Richard Korf and 
colleagues during the week of January 17-23, 1980 (unpublished; specimens 
deposited at CUP) revealed no additional P seaveri specimens. 

Between 1946 and 1953, introduced scale insects decimated the Bermuda 
cedar, killing an estimated 95% (Phillips 1984), thus deforesting the islands and 
altering the ecology of many other Bermudian species in major ways (Thomas 
2004). With no known Plectania seaveri specimens collected since 1940, it 
seems probable that this fungus is now extinct. While it may still occur on 
some of the dead, non-decorticated trunks of Bermuda cedar that now dot the 
Bermudian landscape (and perhaps on the few living Bermuda cedars which 
still persist), it seems likely that the fate of P seaveri may eventually follow that 
of other endemic Bermudian organisms dependent on Bermuda cedar, such as 
the Bermuda cicada (Tibicen bermudiana) and the ochre-banded looper moth 
(Semiothisa ochrifascia) (Sterrer 1998). 

As far as we know, all species of Plectania sect. Donadinia, including 
P. seaveri, associate as saprophytes with coniferous trees. Future collections of 
representatives of this section would help us understand whether this coniferous 
association is taxonomically relevant. 


FINAL CONSIDERATIONS — This study demonstrates that Bulgaria melastoma as 
interpreted by Seaver (1928) needs an in-depth revision. Paden & Tylutki (1969) 
observed that “in Seaver, P milleri will key out to P melastoma (as Bulgaria 
melastoma). We can speculate that, at least in the case of Plectania seaveri, 
Seaver might have misinterpreted the encrusting pigment of the external 
surface, indicating that he was not aware of the true diagnostic characters of 
P. melastoma. In any case, we are convinced that Seaver misapplied the name 
P. melastoma to other specimens, such as one from Puerto Rico (CUP-PR- 
001281, examined as part of the current study), and probably also the Jamaican 
collection cited by Seaver & Waterston (1946). The latter specimen, supposedly 
deposited in NY, could not be located during a recent visit to that herbarium 
(SLG pers. obs., April 2, 2012). Our future studies will attempt to answer these 
lingering questions. 


328 ... Carbone, Agnello & LaGreca 


Acknowledgments 

We are grateful to Mark Whitten and the Electron Microscopy Core Facility at the 
University of Florida (Gainesville, USA) for generously performing the SEM work for 
this study. Dick Korf and Nicolas VanVooren both reviewed a draft of this manuscript; 
we thank them for their helpful comments. Barbara Thiers and Stephen Sinon are 
thanked for their attempts to track down Seaver’s original plates of Bulgaria melastoma 
in the Seaver Collection housed at the Mertz Library, New York Botanical Garden. We 
are thank Ellen Bloch (NY) for providing key information about certain specimens 
cited in this paper, Lucy Klebieko and Mike Bevans (NY) for their helpful assistance 
in scanning the herbarium photograph for Fic. 1, and the Curator of the Herbarium of 
Natural History of Venice (MCVE) for arranging the loan of the examined collections. 


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Plectania seaveri sp. nov. (Bermuda) ... 329 


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ISSN (print) 0093-4666 © 2012. Mycotaxon, Ltd. ISSN (online) 2154-8889 


MY COTAXON 


http://dx.doi.org/10.5248/120.331 
Volume 120, pp. 331-342 April-June 2012 


New species of Entoloma (Basidiomycetes, Agaricales) 
from Kerala State, India 


C.K. PRADEEP’ , K.B. VRINDA’*, SHIBU P. VARGHESE! & T.J. BARON?’ 


"Tropical Botanic Garden & Research Institute, 
Palode, Trivandrum, Kerala, 695562, India 

*Department of Biological Sciences, State University of New York, 
College at Cortland, New York, USA.13045 


*CORRESPONDENCE TO: drckpradeep@hotmail.com 


ABSTRACT — Three new species of Entoloma (E. suaveolens, E. crassum, and E. aurantio- 
quadratum) are described, illustrated, and discussed based on collections made from Kerala 
State, India. 


KEY WORDS — agaric diversity, under-explored, Western Ghats 


Introduction 

Entoloma is one of the largest genera of Agaricales, with more than 1500 
species known worldwide (Noordeloos 1981a). However, vast areas are still 
under-explored, especially in Africa, South America, India, and South East 
Asia (Noordeloos & Morozova 2010). Recent studies of the genus in Kerala 
State, India, by Manimohan et al. (1995, 2002, 2006) documented 39 species, of 
which 26 are new to science, indicating that Entoloma is especially rich in the 
state. While documenting the agaric diversity of Western Ghats of Kerala, the 
authors collected several specimens of Entoloma. Our detailed study of these 
collections has resulted in three new species, which are described, illustrated, 
and discussed here. 


Materials & methods 

Gross morphological descriptions are based exclusively on fresh materials 
collected from Kerala State, India. Color coding follows Kornerup & Wanscher (1978). 
Microscopic characters were studied from hand cut sections of dried basidiomata 
revived in a 3% KOH aqueous solution, stained with 1% Congo red, and examined 
under a Leica DME 1000 compound microscope. The mean quotient (Q) of spore length 
divided by spore width was calculated from measurements of 30 basidiospores; the hilar 


332 ... Pradeep & al. 


appendix was included in the measurement of the spore length. Line drawings were 
made with assistance of an attached drawing tube. All specimens cited were collected 
by the authors unless otherwise indicated. Holotypes are deposited at the Herbarium of 
the Royal Botanic Gardens, Kew (K), and all isotypes and additional materials examined 
are deposited at the Mycological Herbarium of Tropical Botanic Garden and Research 
Institute, Trivandrum (TBGT). 


Taxonomy 


Entoloma suaveolens C.K. Pradeep & K.B. Vrinda, sp. nov. PLATE 1A-B; FIG. 1 
MycoBank MB 564454 
Differs from other blue Entoloma species in combining an appressed squamulose pileus 
and stipe with the lack of hymenial cystidia and the presence of a strong fragrant odor. 
Type -— India, Kerala State, Trivandrum District, Palode, TBGRI campus, 25 Sep 1996, 
Pradeep 3621 (Holotype, K(M) 172393). 


ETyMOLocy -suaveolens refers to the fragrant odor of the basidiomes 


PiLEus 20-53 mm diam., convex with a broad obtuse umbo; surface ink blue, 
dark blue to greyish blue (20F4/21F4/21F8/22F4/21D5) at disc with greyish 
violet to dark blue (18E5/19E4/19E5/19E6) elsewhere, appressed squamulose 
throughout, often cracked to expose the white underlying context in dry 
season; squamules often washed off during heavy rain towards margin to 
appear subsquamulose, dry; margin straight, entire. LAMELLAE adnate, white, 
cream to greyish orange (443/4B4/5A2/5B3/6A2/6B4), < 11 mm wide, close to 
crowded, with lamellulae of different lengths; edge concolourous, entire. STIPE 
35-114 x 3-10 mm, central, cylindric, curved, fistulose to narrowly hollow, 
brittle, tapering up from a subclavate base; surface mostly concolorous with 
pileus or greyish violet (19D5), greyish blue (20D5), appressed squamulose 
throughout; base white with white mycelial mat and mycelial cords. CONTEXT 
white, up to 3 mm, soft. OporR strong, distinct, pleasant, fragrant, similar to 
holy ash. Spore PRINT pink. 

BasIp1osPpores 10-12 x 5-7.5(-8) um, avL = 11, avW = 6.77, Q = 1.3-1.7, 
avQ = 1.65, heterodiametric ovate, 5-7 facets in profile. BAstp1A 32-42.5 x 8.5-14 
um, clavate, 4-spored; sterigmata up to 4.5 um long. LAMELLA EDGE fertile. 
CHEILOCYSTIDIA and pleurocystidia absent. HYMENOPHORAL TRAMA regular; 
hyphae 2.5-16(-28) um wide, thin-walled, hyaline. PILEAL TRAMA interwoven; 
hyphae similar to hymenophoral trama. PILEIPELLIs a trichoderm with clavate 
to cylindro-clavate elements, 62.5-147 x 10-14 um, thin-walled with bluish 
(in water) contents which dissolve in 3% KOH. STIPITIPELLIS a cutis of loosely 
arranged septate thin-walled hyphae, 2.4-4.8 um wide, with pale greyish 
plasmatic contents. CAULOCYSTIDIA in tufts present on the upper part of the 
stipe, 41-67 x 5-9 um, cylindro-clavate, thin-walled with pale brown vacuolar 
contents. CLAMP CONNECTIONS and oleiferous hyphae present in all parts. 


Entoloma spp. nov. (India) ... 333 


1S) 


Fic. 1: Entoloma suaveolens. A, basidia; B, basidiospores; 
C, trichodermial elements; D, caulocystidia. Scale bars = 10 um. 


334 ... Pradeep & al. 


HABIT & HABITAT — Solitary, scattered on soil among decayed litter in 

evergreen forest, June-December. 
ADDITIONAL SPECIMENS EXAMINED — INDIA, KERALA STATE, TRIVANDRUM DISTRICT, 
Palode, TBGRI campus: 16 Sep 1997, TBGT 3887; 15 Oct 2001, TBGT 6770; 8 Jun 2005, 
TBGT 8907; 4 Nov 2005, TBGT 9425; 17 Jul 2007, TBGT 10427; 25 Jul 2008, TBGT 
11513; 30 Jul 2008, TBGT 11554; 16 Dec 2009, TBGT 13058; Wayanap District, 
Nadavayal: 27 Sep 2007, TBGT 10575 (all at TBGT). 
Discussion — Entoloma suaveolens is characterized by the unique 
combination of medium to large bluish terrestrial tricholomatoid basidiomes, 
convex umbonate appressed squamulose pileus and stipe, adnate pink 
lamellae, heterodiametric ovate basidiospores, absence of hymenial cystidia, 
trichodermial pileipellis, abundant caulocystidia, clamp connections present 
in all hyphae, and strong fragrant odor. 

The present collections invite comparison with a number of blue Entoloma 
species described from various regions of the world by different workers. 
Entoloma rugosopruinatum Corner & E. Horak, originally described from Sabah 
(Horak 1980) and recently recorded from Kerala (Manimohan et al. 1995), 
differs by its violaceous purple finely tomentose pileus, presence of cylindro- 
clavate cheilocystidia, and subfarinaceous odor. Entoloma divum Corner & 
E. Horak is distinguished by a deep blue pruinose pileus, palisadic pileipellis, 
and lack of any odor. Three other comparable blue species —E. simillimum 
Corner & E. Horak, E. egregium E. Horak, E. marinum Corner & E. Horak— are 
easily separated by their velutinous pileus, presence of cheilocystidia, and lack 
of any distinctive odor. The European taxa E. chalybeum (Pers.) Noordel. and 
E. chalybeum var. lazulinum (Fr.) Noordel. (Noordeloos 1992) are distinguished 
by their depressed velutinous pileus, heterodiametrical 5-9-angled spores, sterile 
lamella edge, a cutis pileipellis, and absence of clamps. Entoloma bloxamii (Berk. 
& Broome) Sacc. and E. nitidum Quél. (Horak 1973, 1978, 1980; Noordeloos 
1992), though comparable in gross morphology and pigmentation, vary 
markedly in their smooth glabrous subviscid pileus (pileipellis an ixocutis) and 
distinctive farinaceous odor. Entoloma dichroum (Pers.) P. Kumm. (Noordeloos 
1992) differs in its violaceous purple pileus, heterogeneous lamella edge, and 
unpleasant odor. The recently described E. eugenei Noordel. & O. Morozova 
(Noordeloos & Morozova 2010) from Russia differs in its velvety deep blue 
pileus, sterile lamellae edge with cylindrical, narrowly lageniform or irregularly 
shaped cheilocystidia, and mild spicy odor. 

Leptonia carnea Largent, a rather uncommon species restricted to coastal 
and northern California, is characterized by a blue black densely appressed 
fibrillose squamulose pileus and stipe, blue—violet-black lamella edge, cuticular 
pileipellis, and distinctive farinaceous odor and taste (Largent 1977); L. carnea 
is further distinguished by the insoluble blue pigment, contrasting with the 
readily KOH-soluble pigment in E. suaveolens. 


Entoloma spp. nov. (India) ... 335 


Two recently described blue species from Kerala State, E. griseolazulinum 
Manim. & Noordel. and E. indoviolaceum Manim. & Noordel. (Manimohan 
et al 2006), clearly differ in their velutinous pileus, presence of cheilocystidia, 
large heterodiametric spores, and lack of any distinct odor. 

Tricholomatoid habit, convex umbonate bluish pileus, trichodermial 
pileipellis, and presence of clamp connections place this species in subg. 
Leptonia sect. Leptonia (Noordeloos 1981a). 


Entoloma crassum C.K. Pradeep & K.B. Vrinda, sp. nov. PLATE 1C-D; Fic. 2 
MycoBank MB 564455 
Differs from Entoloma flavidum in its glabrous creamy to yellow white pileus, lack of 
cheilocystidia, and strong, nauseating odor. 
Type — India, Kerala State, Trivandrum District, Palode, TBGRI campus: 7 Oct 2004, 
Pradeep 8154 (Holotype, K(M) 172394). 


ETYMOLOGY —crassum refers to the thick, stout basidiomes 


Basip1oMsEs thick, fleshy, robust, tricholomatoid. PiLeus 45-160 mm diam., 
convex to planoconvex with a broad obtuse umbo, becoming uplifted in old 
ones; surface uniformly creamy white to yellowish white (442/443) when young 
and yellowish white to brownish orange (442/443/3B3/3C3/4B2/ 4B3/4B4) with 
brownish disc (48B3/4B4/4C4/5C2/5C3/5D3/5D4/5E3/5E4/6E4) in mature ones, 
moist, smooth, glabrous, not hygrophanous; margin incurved when young 
becoming straight, wavy, folded, entire to incised. LAMELLAE broadly adnate to 
subdecurrent, yellowish white to greyish red (442/5A2/6A2/5B3/6C6/7B4), < 20 
mm wide, close to crowded with lamellulae of 3-4 lengths; edge concolourous, 
entire. STIPE 35-150 x 10-25 mm, central (often excentric in some specimens), 
cylindric (rarely compressed), twisted, thick, fibrous, solid, equal, with a slightly 
broad base; surface white, cream, ivory (483) turning brownish orange (5c3), 
hair brown (584), greyish brown (6D3), brownish grey (6£2) on handling, 
fibrillose striate, smooth. Basal white mycelial mat present in some specimens. 
CONTEXT white, < 11 mm thick, soft, fleshy. ODor strong, unpleasant, pungent, 
nauseating. SPORE PRINT cinnamon to reddish brown (6D6/8D6). 
BASIDIOSPORES (8—)9-10 x (6-)6.5-7.5 um, avL = 9.3, avW = 6.8, Q = 1.25-1.50, 
avQ = 1.34, heterodiametric ovate with 5-6 facets in profile. Basidia 45-58.5 
x 7.5-10 um, clavate, 4-spored. LAMELLA EDGE fertile, CHEILOCYSTIDIA and 
pleurocystidia absent. HYMENOPHORAL TRAMA regular; hyphae 2.5-14 um 
wide, thin-walled, hyaline. PILEAL TRAMA interwoven; hyphae similar to that of 
hymenophoral trama. PILEIPELLIs a cutis passing to a trichoderm at the centre, 
elements 15-38 x 4-8 um, clavate, cylindro-clavate to strangulated, thin- 
walled, hyaline. STIPITIPELLIs a cutis of longitudinally parallel hyphae, 2.5-5 
uum wide, thin-walled, hyaline. CauLocystip1a abundant throughout the stipe 
surface, 25-80 x 6-9 um, lageniform or narrowly utriform, cylindric, cylindro- 


336 ... Pradeep & al. 


clavate, subutriform, strangulated, flexuous, often with a short subobtuse apex, 
thin-walled with intracellular granular contents. CLAMP CONNECTIONS and 
OLEIFEROUS HYPHAE present. 
HasitT & Hasitat — Solitary, scattered or in groups on soil among litter in 
evergreen forest or under reeds in sandy soil on riverbanks, April-December. 
ADDITIONAL SPECIMENS EXAMINED — INDIA, KERALA STATE, TRIVANDRUM DISTRICT, 
Palode, TBGRI campus: 21 Oct 1995, TBGT 2758; 19 May 1997, TBGT 3858; 13 May 
1999, TBGT 4659; 19 Jun 2000, TBGT 5072; 21 Jun 2000, TBGT 5081; 27 Jun 2001, 
TBGT 5351; 29 Apr 2002, TBGT 5502; 7 Jul 2005, TBGT 9064; 23 Sep 2005, TBGT 
9278; 17 Jun 2008, TBGT 11122; 25 Jun 2008, TBGT 11237; 30 Sep 2008, TBGT 12064; 
4 Nov 2008, TBGT 12189; 11 Jun 2009, TBGT 12577; 16 Jun 2009, TBGT 12606; 29 
Jun 2009, TBGT 12685; 16 Sep 2009, TBGT 12918; 23 Sep 2009, TBGT 12940; 14 Oct 
2009, TBGT 12980; 23 Oct 2009, TBGT 13012; 25 Nov 2009, TBGT 13110; 11 Dec 2009, 
TBGT 13128; 6 Jul 2010, TBGT 13373; 4 Aug 2011, TBGT 13768; Kallar: 10 Jul 2007, 
TBGT 10411 (all at TBGT). 
Discuss1on — Entoloma crassum is a very remarkable species characterized by 
large robust tricholomatoid basidiomata, a convex umbonate smooth glabrous 
yellowish white pileus with a brownish disc, broadly adnate to subdecurrent 
lamellae, a fibrillose striate white stipe that bruises brown, strong unpleasant 
nauseating odor, heterodiametric spores, fertile lamella edge, trichodermial 
pileipellis, caulocystidia that are abundant over the entire stipe surface, and 
presence of clamp connections in all hyphae. 

The most closely related entolomas with large tricholomatoid yellowish 
white basidiomes are compared and discussed here. Despite its macroscopic 
and microscopic similarities, E. flavidum (Massee) Corner & E. Horak (Horak 
1980) differs from E. crassum in its white subtomentose to floccose pileus that 
yellows with age, hollow fibrillose-striate non-yellowing white stipe, cylindric 
to subclavate cheilocystidia arrayed on a sterile lamella edge, a cuticular 
pileipellis, sour odor, and absence of caulocystidia. Among similar species that 
differ by possessing an ixocuticular pileipellis, E. sinuatum (Bull.) P. Kumm. 
(Noordeloos 1981b) is further distinguished by its viscid pale ochraceous 
pileus, characteristically yellow lamellae, and small isodiametric spores; 
E. niphoides Romagn. ex Noordel. (Largent 1994) is separated by its snow-white 
pileus and stipe and fruity odor; E. moserianum Noordel. (Noordeloos 1983) 
is distinguished by its typically yellow spotted pileus, lamellae, and stipe and 
sterile lamella edge; E. albomagnum G.M. Gates & Noordel. and E. cretaceum 
G.M. Gates & Noordel. (Gates & Noordeloos 2007) disagree by their pure white 
pileus and small spores; and E. sepium (Noulet & Dass.) Richon & Roze and 
E. saundersii (Fr.) Sacc. (Breitenbach & Kranzlin 1995) differ mainly in their 
silky white pileus, farinaceous odor, and lack of caulocystidia. 

The large tricholomatoid habit, nonhygrophanous convex umbonate smooth 
pileus, adnate decurrent lamellae, regular hymenophoral trama, and abundant 


Entoloma spp. nov. (India) ... 337 


Fic. 2: Entoloma crassum. 
A, basidia; B, basidiospores; C, trichodermial elements; D, caulocystidia. Scale bars = 10 um. 


338 ... Pradeep & al. 


clamps indicate that the species belongs to subg. Entoloma sect. Entoloma 
(Noordeloos 1981a). 


Entoloma aurantioquadratum C.K. Pradeep & K.B. Vrinda, sp. nov. 
MycoBAank MB 564456 PLATE 1E-F; FIG. 3 


Differs from Entoloma talisporum and E. gracilius in the absence of clamp connections, 
from E. hyalodepas in its large quadrate basidiospores, and from E. albogracile in the 
absence of hymenial cystidia. 


Type — India, Kerala State, Trivandrum District, Palode, TBGRI campus: 11 Nov 2009, 
Shibu 13048 (Holotype, K(M) 172395). 


ETYMOLOGY — aurantioquadratum refers to the orange basidiome with quadrate 

spores 
PrLEus 10-30 mm diam., convex to applanate with a small shallow depression 
at centre, often uplifted in old ones; surface orange white to brownish orange 
(5A2/5A3/5B2/5B3/5C3), with hair brown to greyish brown (5£4/7D3/7E3) 
centre or even paler towards margin, dry, velvety at disc (under a lens), elsewhere 
smooth, glabrous, nonhygrophanous; margin straight to uplifted in old ones, 
wavy, entire to incised, pellucid striate when wet. LAMELLAE adnexed, orange 
white to pinkish white (542/7A2), up to 4 mm wide, close with lamellulae of 
different lengths; edge concolourous to the sides, entire. STIPE 20-50 x 1-3 
mm, central, cylindric, twisted, hollow, equal, curved, often narrowly tapering 
up from a slightly broad base; surface white turning brown on handling or 
bruising, pruinose in the upper part (under the lens) elsewhere smooth and 
glabrous. CONTEXT thin, concolourous to pileus, soft. Odor none. 

BASIDIOSPORES (9-)9.5-12.5(-13) x (6-)6.5-8.5(-9.5) um, avL = 10.8, avW 
= 7.6, Q = 1.05-1.7, avQ = 1.4, quadrate in profile. Basidia 23-36 x 9-12.5 um, 
clavate, 4-spored. LAMELLA EDGE fertile, CHEILOCystTID1A and pleurocystidia 
absent. HYMENOPHORAL TRAMA regular, 2-12 um wide, thin-walled, hyaline. 
PILEAL TRAMA interwoven; hyphae similar to hymenophoral trama. PILEIPELLIS 
a cutis with transitions towards a trichoderm at centre, elements 19.5-65 x 
7.5-11 um, clavate to cylindro-clavate, thin-walled with brown plasmatic 
intracellular contents. STIPITIPELLIS a cutis of loosely arranged, septate, thin- 
walled, 2-4 um wide, hyaline hyphae. CAULOCysTIDIA in groups on the upper 
part, 14-32 x 4.5-10.5 um, clavate, cylindro-clavate with septate base, thin- 
walled, hyaline. CLAMP CONNECTIONS absent. OLEIFEROUS HYPHAE present. 
HaBiT & HaBiTaT — Scattered on forest floor in tropical evergreen forest, 

July-November. 

ADDITIONAL SPECIMENS EXAMINED — INDIA, KERALA STATE, TRIVANDRUM DISTRICT, 

TBGRI campus: 10 Nov 2009, TBGT 13043; 15 Jul 2011, TBGT 13685; 11 Aug 2011, 

TBGT 13781 (all at TBGT). 
Discussion — Entoloma aurantioquadratum is a typical member of subg. 
Inocephalus due to its nonhygrophanous nonstriate pileus and quadrate spores. 


Entoloma spp. nov. (India) ... 339 


Fic. 3: Entoloma aurantioquadratum. 
A, basidia; B, basidiospores; C, trichodermial elements; D, caulocystidia. Scale bars = 10 um. 


340 ... Pradeep & al. 


PLATE 1: A-B, Entoloma suaveolens; C-D, E. crassum; 
E-E, E. aurantioquadratum. Scale bars: A-B, E-F = 10 mm; C-D = 20 mm. 


Entoloma spp. nov. (India) ... 341 


The combination of small collybioid basidiomes with pale orange pileus, white 
stipe turning brownish, large spores, fertile lamellae edge, cuticular pileipellis 
with transitions towards a trichoderm, presence of caulocystidia, and lack of 
clamp connections are diagnostic for this species. 

A comprehensive literature search (Horak 1976, 1977, Hesler 1967, Pegler 
1986, Noordeloos 1987, 1992, Noordeloos & Hausknecht 2007, Manimohan 
et al. 1995, 2006) revealed that although some species are comparable, none 
matches E. aurantioquadratum exactly. Entoloma talisporum Corner & E. 
Horak differs in its yellowing white fibrillose pileus, smaller cuboid spores, and 
presence of clamp connections. Entoloma hyalodepas (Berk. & Broome) E. Horak 
is distinguished by its depressed to infundibuliform ivory yellow or ochraceous 
buff pileus, decurrent lamellae, large pentagonal spores, and cuticular pileipellis 
(Baroni & Lodge 1998). Entoloma albogracile E. Horak has a convex-umbonate 
papillate white to yellowish pileus, cylindrical to subclavate cheilocystidia, and 
cuboid to tetrahedral spores. Entoloma gracilius E. Horak can be separated 
by its small fragile basidiomes, yellowish green tinted pileus, broadly adnate 
lamellae, smaller cuboid spores, and presence of clamp connections. 


Acknowledgments 

The authors are grateful to Drs. Alfredo Justo (Biology Department, Clark 
University, Worcester, USA) and Laura Guzman-Davalos (Departamanto de Botanica 
y Zoologia, Universidad de Guadalajara, Mexico) for their pre-submission reviews and 
suggestions. 


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Breitenbach J, Kranzlin F 1995. Fungi of Switzerland 4: Agarics 2nd part. Mykologia Lucerne, 
Switzerland. 

Gates GM, Noordeloos ME. 2007. Preliminary studies in the genus Entoloma in Tasmania - I. 
Persoonia 19: 157-226. 

Hesler LR. 1967. Entoloma in southeastern North America. Beihefte Nova Hedwigia 23: 1-196. 

Horak E. 1973. Fungi Agaricini Novazeylandiae. 1. Entoloma Fr. and related genera. Beihefte Nova 
Hedwigia 43: 1-86. 

Horak E. 1976. On cuboid species of Entoloma. Sydowia 28: 171-236. 

Horak E. 1977. Additions to “On cuboid-spored species of Entoloma”. Sydowia 29: 289-299. 

Horak E. 1978. Entoloma in South America. 1. Sydowia 30: 40-111. 

Horak E. 1980. Entoloma (Agaricales) in Indomalaya and Australasia. Beihefte Nova Hedwigia 65: 
1-352; 

Kornerup A, Wanscher JH. 1978. Methuen handbook of colour. 3rd ed. Methuen, London. 

Largent DL. 1977. The genus Leptonia on the Pacific coast of the United States. Bibliotheca 
Mycologica 55: 1-309. 

Largent DL. 1994. Entolomatoid fungi of the Western United States and Alaska. Mad River Press, 
Eureka, CA. 


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Manimohan P, Vijaya Joseph A, Leelavathy KM. 1995. The genus Entoloma in Kerala State, India. 
Mycological Research 99: 1083-1097. http://dx.doi.org/10.1016/S0953-7562(09)80777-6 

Manimohan P, Leelavathy KM, Noordeloos ME. 2002. Three new species of Entoloma from Kerala 
State, India. Persoonia 17: 625-630. 

Manimohan P, Noordeloos ME, Dhanya AM. 2006. Studies on the genus Entoloma (Basidiomycetes, 
Agaricales) in Kerala State, India. Persoonia 19: 45-93. 

Noordeloos ME. 1981a. Introduction to the taxonomy of the genus Entoloma sensu lato (Agaricales). 

Persoonia 11: 121-151. 

Noordeloos ME. 1981b. Entoloma subgenera Entoloma and Allocybe in the Netherlands and adjacent 

regions with a reconnaissance of their remaining taxa in Europe. Persoonia 11: 153-256. 

Noordeloos ME. 1983. Studies in Entoloma — 9. On two new European species in section Entoloma. 

Sydowia 36: 208-212. 

Noordeloos ME. 1987. Entoloma (Agaricales) in Europe. Beihefte Nova Hedwigia 91: 1-417. 

Noordeloos ME. 1992. Entoloma s.l. Fungi Europaei, Saronno, Italy. 

Noordeloos ME, Hausknecht A. 2007. The genus Entoloma (Basidiomycetes, Agaricales) of the 

Mascarenes and Seychelles. Fungal Diversity 27: 111-144. 


Noordeloos ME, Morozova OV. 2010. New and noteworthy Entoloma species from the Primorsky 
Territory, Russian Far East. Mycotaxon 112: 231-255. http://dx.doi.org/10.5248/112.231. 
Pegler DN. 1986. Agaric flora of Sri Lanka. Kew Bulletin Additional Series 12: 1-519. 


ISSN (print) 0093-4666 © 2012. Mycotaxon, Ltd. ISSN (online) 2154-8889 


MY COTAXON 


http://dx.doi.org/10.5248/120.343 
Volume 120, pp. 343-352 April-June 2012 


A new species of Infundichalara from pine litter 


ONDREJ KOUKOL 


Department of Botany, Charles University in Prague, 
Bendtska 2, CZ-128 01 Prague, Czech Republic 
CORRESPONDENCE TO: ondrej.koukol@natur.cuni.cz 


ABSTRACT — A new species of Infundichalara (anamorphic Helotiales), a hitherto monotypic 
genus, is described from pine needle litter. Infundichalara minuta sp. nov. forms two 
conidiophore types. Erect penicillate conidiophores producing catenulate non-septate 
hyaline conidia form more frequently than Chalara-like conidiophores with funnel-shaped 
collarettes produce wedge-shaped phialoconidia. Although its morphological characters 
correspond more with Xenopolyscytalum, a three-region DNA analyses (ITS, 28S rDNA and 
EF-1la) of Infundichalara, Xenopolyscytalum, and related Chalara species in the Helotiales 
confirm the phylogenetic position of the new species within Infundichalara. It differs from 
I. microchona by smaller phialides and wedge-shaped conidia. 


Key worps — hyphomycete, dimorphic conidiophores, litter saprotroph 


Introduction 

Scots pine (Pinus sylvestris L.) needles in litter host several microfungal 
species producing straight erect penicillate conidiophores with catenulate 
hyaline conidia. Polyscytalum fecundissimum Riess and P. pini P.M. Kirk & 
Minter produce uniseptate conidia from denticulate conidiogenous cells 
growing on a straight, erect conidiophore (Kirk 1983). Hormiactella asetosa 
Hol.-Jech., differing from Polyscytalum by conidia with rounded ends and dark 
grey colonies (Holubova-Jechova 1978), predominantly colonizes bark but 
may also be found on needles in the litter (Koukol 2007). A monotypic genus 
Xenopolyscytalum Crous was described for X. pinea Crous to accommodate 
two strains isolated from pine needles (Crous & Groenewald 2010). Unlike 
Polyscytalum and Hormiactella representatives, X. pinea forms white tufts on 
needles and is characterized by aseptate conidia with somewhat darkened hila 
and a Chalara-like synanamorph. The synanamorph produces phialides with a 
flaring collarette that differs from the tubular collarette found in Chalara (Corda) 


344 ... Koukol 


Rabenh. (Nag Raj & Kendrick 1975). The only Chalara species characterized by 
a funnel-shaped collarette, C. microchona W. Gams, was recently placed into a 
new genus, Infundichalara Réblova & W. Gams (Réblova et al. 2011). 

I have isolated one fungus with dimorphic (penicillate/Chalara-like) 
conidiophores from needles of several pine species from various parts of Europe. 
These isolates are morphologically similar to both Xenopolyscytalum (abundant 
production of penicillate conidiophores) and Infundichalara (Chalara-like 
conidiophores with funnel-shaped collarettes) but obviously represent a 
distinct species. Both morphological and molecular criteria were considered to 
accommodate the species in a genus reflecting evolutionary relationships. 


Materials & methods 


Collection and isolation 

Cultures were isolated from needles of Scots pine (Pinus sylvestris), Swiss stone pine 
(P. cembra L.), Siberian pine (P. sibirica Du Tour), and Siberian dwarf pine (P pumila 
(Pallas) Regel) in the litter. Needles were sampled in pure or mixed pine forests in the 
NP Bohemian Switzerland (Czech Republic), along the rivers Timpton (Republica 
Sakha) and Vyerchni Sakujan (Zabaykalskaya Oblast) in Russia, and in the Windachtal 
Valley, Tyrol (Austria). Surface sterilisation of needles and cultivation conditions were 
identical to those described in Koukol (2010). Culture characteristics were observed on 
2% malt agar (2MA, final sucrose content 2% w/v, 18 g agar, 1 | distilled water) prepared 
from brewer's wort (Staropramen Brewery, Prague, Czech Republic), potato carrot agar 
(PCA), oatmeal agar (OA), and potato-dextrose agar (PDA), all prepared from fresh 
ingredients according to Fassatiova (1986). Agar plates were maintained at laboratory 
temperature (22-25°C). For microscopy and measurements, conidiophores with conidia 
were mounted in Melzer’s reagent and examined with phase or differential interference 
contrast (using an Olympus BX51 microscope with digital camera, measured using 
Quick Photo software). Pictures were further edited in Adobe Photoshop®. Microscopic 
measurements are reported as the mean + standard deviation of 30-50 measurements 
with the extremes given in parentheses. 

The holotype specimen is deposited in the PRM (National Museum, Prague, Czech 
Republic) and living cultures are maintained in the CCF (Culture Collection of Fungi, 
Prague, Czech Republic). 


DNA extraction, amplification and sequencing 

DNA was isolated from cultures grown on 2MA using the ZR Fungal/Bacterial DNA 
kit (Zymo Research, Orange, CA, USA). DNA analyses were made with sequences from 
the regions ITS1+5.8S+ITS2 (referred to it as the ITS rDNA) and 28S rDNA together 
with the gene coding for the translation elongation factor 1 a (EF-la). Amplification 
and sequencing were performed according to Koukol (2011) with the exception that 
only the shorter part of EF-1a was amplified with primer pairs 983F a 1567R (Rehner 
& Buckley 2005). New sequences generated in this study from newly isolated strains 
are listed in TaBLE 1. Sequences of ITS and 28S rDNA of X. pinea were obtained from 


Infundichalara minuta sp. nov. ... 345 


TABLE 1. Accession numbers of new sequences generated in this study. 


SPECIES / CULTURE ITS 28S RDNA EF-1a 
Infundichalara minuta 
CCF4156, ex-type HE603986 HE603981 HE603978 
CCF4157 HE603987 HE603982 HE603977 
CCF4158 HE603988 HE603983 HE603976 
CCF4159 HE603989 HE603984 — 
CCF4160 HE603990 HE603985 — 
Xenopolyscytalum pinea 
CPC14225, ex-type — _— HE603980 
CPC14234 — — HE603979 


GenBank. The remaining sequences of Chalara spp., Cistella acuum (Alb. & Schwein.) 
Svréek, and Infundichalara microchona (W. Gams) Réblova & W. Gams originated in 
Koukol (2011). 


Phylogenetic analyses 

All markers were aligned using MUSCLE (Edgar 2004) and adjusted manually 
in BioEdit, v. 4.7.1 (Hall 1999) to maximize similarities. The dataset consisted of 32 
sequences and comprised 1322 characters with 272 parsimony-informative sites. 
Phylogenetic analyses were conducted using MEGA v. 5 (Tamura et al. 2011), PhyML 
v. 3.0 (Guindon et al. 2010) and MrBayes v. 3.1.2 (Ronquist & Huelsenbeck 2003). For 
the Bayesian analyses, General Time Reversible model with invariant sites and a gamma 
rate distribution (GTR+I+G) was used due to its maximum complexity. The dataset was 
divided to three partitions (ITS, 28S rDNA and EF-1a). The proportion of variable sites, 
rate frequencies and the gamma shape were estimated separately for the three partitions. 
Markov chains were initiated from a random tree and were run for 6,000,000 generations; 
samples were taken every 100th generation. Posterior probabilities (PP) were used as a 
Bayesian branch support on the consensus trees. In addition, bootstrap branch support 
(BS) values were estimated using the Maximum-Likelihood (ML) analyzes with 10,000 
bootstrap replicates (MEGA 5.0, PhyML v. 3.0). The tree was rooted with Tryblidiopsis 
pinastri (Pers.) P. Karst. (AFTOL-ID 1319, sequences of 28S rDNA DQ470983, EF-1la 
DQ471106). 


Results 

Five strains isolated during this study (CCF4156-4160) formed a strongly 
supported group sister to I. microchona (CBS175.74 and CBS889.73). The isolate 
B282 obtained from pine needles in Tyrol (Austria) clustered with X. pinea 
(Fic. 1). It most probably belonged to X. pinea, but was not morphologically 
observed, because it was lost due to contamination after DNA extraction. 
Xenopolyscytalum pinea (CPC14225 and CPC14234) clustered with low support 
with Cistella acuum (CCF3970). 


346 ... Koukol 


100/99 , C. hyalocuspica CCF3976 
C. hyalocuspica CCF3975 
C. affinis CBS562.77 
C. pseudoaffinis CBS261.75 
159 100/- | C. pseudoaffinis CCF3979 
C. microspora CBS131.74 
C. microspora CCF3980 
135+ C. microspora CCF3981 
100/98 - C. holubovae CCF3978 


C. holubovae ZK68 
Cistella acuum CCF3970 


99/100 


100/100 


61/- 86/74, X. pinea CPC14225 
an 99/62! | X pinea CPC14234 
X pinea B2S2 


100/71 C. recta CCF 3936 
C. recta S17-2 
79/82 | C. longipes CCF3973 
C. longipes CCF3972 
92/88 C. longipes CBS264.94 
56/- C. longipes CBS411.76 
100/99 C. longipes CCF3974 
70184 | ©. longipes ZK66 
C. piceae-abietis CCF3983 
100/84 C. piceae-abietis CCF3982 
81/100 — |. microchona CBS175.74 
| microchona CBS889.73 
/5A |. minuta CCF4160 
|. minuta CCF4159 
I. minuta CCF4156 
|. minuta CCF4157 


100/67 L |. minuta CCF4158 
Tryblidiopsis pinastri AFTOL-ID 1319 


96/- 


100/63 100/- 


SS 
0.1 


Fic. 1. Phylogenetic relationships of Infundichalara minuta and selected members of Helotiales 
derived from ITS, 28S rDNA and EF-la gene regions using ML and BA analyses. Genera are 
abbreviated as C. = Chalara, X. = Xenopolyscytalum, and I. = Infundichalara. Ex-type sequences are 
in bold. Bootstrap values > 50% are indicated along nodes. Thick lines show PP > 0.95. The tree was 
rooted with Tryblidiopsis pinastri. 


Taxonomy 


Infundichalara minuta Koukol, sp. nov. Figs 2, 3 a,b 
MycoBank MB 563538 
Differs from Infundichalara microchona by its smaller Chalara-like phialides 
producing wedge-shaped conidia with truncate base and synanamorph with penicillate 
conidiophores, ramoconidia, and subcylindrical conidia. 


Infundichalara minuta sp. nov. ... 347 


Fic. 2. Infundichalara minuta: colonial and microscopic morphology. a-d, Morphological 
variation on 2MA after 30 d incubation at 22-25°C; e, penicillate conidiophore with ramoconidia. 
f-h, Chalara-like conidiophores (f, intercalary; h, terminal); i, conidia produced on penicillate 
conidiophores; j, conidia produced from Chalara-like conidiophores—single, short chain, 
and cluster. (a,e,g,i,j = ex-type strain CCF4156; b = CCF4160; c,fjh = CCF4158; d = CCF4159. 
Bars = 20 um.) 


TyPE: Czech Republic, Bohemian Switzerland National Park, Doubice, Tokan, forest 
of P. sylvestris on sandstone rocks, 50°53.3'N 14°24.8'E, 450 m asl., on needles in the 
litter of P. sylvestris, 11 Dec 2006, leg. O. Koukol (Holotype, PRM899344; ex-type strain 
CCF4156). 


ETYMOLOGY: minutus = referring to the minute Chalara-like conidiophores. 


VEGETATIVE MYCELIUM consisting of smooth or warted, branched, septate, 
hyaline to pale brown hyphae 1.5-3 um diam. ConipiopHoREs dimorphic. 
PENICILLATE CONIDIOPHORES erect, producing branched, catenulate conidia 
visible as white tufts on needles and in culture, pale brown, 3-5-septate, < 40 
um tall, 3-3.5 um wide. CONIDIOGENOUS cells apical, hyaline to pale brown, 
smooth, 9.5-13.5(-15.5) x 1.5-3.5 um, proliferating sympodially, usually giving 
rise to 3 ramoconidia. RaMoconrpzI¢ hyaline, smooth, aseptate, cylindrical to 
subcylindrical, (8—)9-13(-16) x 1.5-3 um. Conip1A subcylindrical, hyaline, 
smooth, aseptate, occurring in unbranched dispersible chains, ends with a 
flattened, slightly protruding scar, 0.8 um wide, (5-)7-9.5(-11.5) x 1.5-2.5 um. 
CHALARA-LIKE CONIDIOPHORES produced less frequently, formed either by 
conidiogenous cell on the mycelium or by conidiogenous cell on 1-4 basal cells, 


348 ... Koukol 


that are erect, cylindrical, unbranched, pale brown, smooth, 4-10 um tall (rarely 
< 50 um), 2.5-4 um wide. CONIDIOGENOUS CELLS phialidic, hyaline to slightly 
pigmented, mostly formed terminally or intercalary directly on mycelium or 
on one supporting cell, subconical to lageniform, (8-)9.5-13.5(-20) x 3-4.5 
um, collarette mostly funnel-shaped, rarely cylindrical, 2.5-5 x 1.5-2.5 um. 
ConipiA wedge-shaped with truncate base, hyaline, smooth, 3-4(-5) x 2-3 
um, forming either short chains or clusters on top of the phialide. 

Cotonts variable, different strains produced different morphologies in 
the dark, after 1 mo at 22-25°C (Fic. 2a-d). Colonies on 2MA erumpent in 
the centre, with dense aerial mycelium, surface dark gray to brown with pale 
margin; reverse pale gray to dark brown, reaching 8-28 mm diam. On PDA 
aerial mycelium developed, colonies gray to pale brown, protuberant in the 
centre, reaching 10-26 mm diam. On OA dendritic, slimy, aerial mycelium 
only at margins, surface dark gray to brown, reaching 17-22 mm diam. On 
PCA gray to pale brown, immersed, with protuberant centre, reaching 8-33 
mm diam. The strain CCF4157 produced pale white immersed mycelium on 
OA, 2MA and PCA and diffused violet pigment into agar on PDA and reached 
< 40 mm diam. on each agar medium tested. Most intensive sporulation of all 
strains was observed on 2MA. 

EcoLocy & DISTRIBUTION — colonizes needles in the litter and humus of 
P. sylvestris, P. cembra, P. sibirica, and P. pumila, in Czech Republic, Austria, 
Russia (this study) and Sweden (concluded from Lindahl et al. 2010). 

ADDITIONAL SPECIMENS EXAMINED: AUSTRIA, TyRroi, Windachtal Valley, sparse P. 
cembra forest above river, 46°57.57'N 11°02.61'E, 1828 m asl., on P. cembra needles 
in litter, 25 Jun 2010, leg. O. Koukol (PRM899346, living culture CCF4160). RUSSIA, 
REPUBLICA SAKHA, sparse growth of P sibirica within Larix gmelinii (Rupr.) Rupr. on 
the right bank of river Timpton near the branching with Ojumrak, 57°11.3'N 126°6.6'E, 
550 m asl., on P. sibirica needles in litter, 4 Aug 2010, leg. D. Svoboda (living culture 
CCF4157); sparse growth of P sibirica within L. gmelinii on the right bank of river 
Timpton near the branching with Bolshoy Ulimakh 58°37.6’N 127°1.1'E, 270 m asl., 
P. sibirica needles in litter, 10 Aug 2010, leg. D. Svoboda (PRM899345, living culture 
CCF4158); ZABAYKALSKAYA OBLAST, P. pumila forest on the right bank of river 


Vyerchni Sakujan, 56°48.5'N 118°6.9’E, 766 m asl., on P pumila needles in litter, 12 Aug 
2010, leg. D. Svoboda (living culture CCF4159). 


Discussion 

Infundichalara minuta shares morphological characteristics with both 
Infundichalara and Xenopolyscytalum. It produces hyaline to slightly 
pigmented phialides with funnel shaped collarettes and conidia that are 
truncate on the base and rounded at the apex (Fic. 2f-h,j), which is consistent 
with Infundichalara (Réblova et al. 2011). On the other hand, Chalara-like 
phialides and a Polyscytalum-like synanamorph with penicillate conidiophores 
(Fra. 2e,i) forming white tufts on the substrate are typical for Xenopolyscytalum 


Infundichalara minuta sp. nov. ... 349 


Fic. 3. Infundichalara minuta (ex-type strain CCF4156): a, Penicillate conidiophore and conidia; 
b, Chalara-like conidiophores and conidia. Xenopolyscytalum pinea (ex-type strain CPC14225): 
c, penicillate conidiophore and conidia. (Bar = 20 um). 


(Crous & Groenewald 2010). Molecular data confirmed placement of the new 
species in Infundichalara, despite the closer morphological similarity with 
Xenopolyscytalum. Obviously, the presence of a Polyscytalum-like synanamorph 
is not phylogenetically significant at the genus level but may be used for 
recognizing the species (e.g., C. holubovae Koukol) that form typical Chalara 
phialides and produce a synanamorph with fusiform conidia (Koukol 2011). 
The previously monotypic Infundichalara and Xenopolyscytalum represented 
by I. microchona and X. pinea respectively may be distinguished from I. minuta 


350 ... Koukol 


based on morphology. Infundichalara microchona produces larger phialides 
(18-35 x 2.5-4.0 um) but with shorter collarettes. The similarly sized 
phialoconidia of I. minuta and I. microchona differ in shape — wedge-shaped 
in I. minuta but clavate in I. microchona (Gams & Holubova-Jechova 1976). 
Infundichalara minuta phialides with the rather narrow collarette typical of 
Chalara s.str. are considered exceptional. Such variation has been noted as well 
for I. microchona (strain CBS125.74) with slightly longer collarettes (to 4 um 
deep; Gams & Holubova-Jechova 1976). Vice versa, molecular data support 
C. longipes (strain CBS867.73), which produces phialides with both types of 
collarettes, in Chalara (Koukol 2011). Xenopolyscytalum pinea is distinguished 
by shorter conidia formed on penicillate conidiophores (3-4(-7) x 1.5(-2) 
um (Fic. 3c), larger Chalara-like phialides (15-25 x 2-3 um), and cylindrical 
phialoconidia. Culture characteristics also differ, for X. pinea grew more quickly 
on all tested media (Crous & Groenewald 2010). 

In this study, only freshly isolated cultures of I. minuta were surveyed. Pure 
cultures were obtained by picking conidia from penicillate conidiophores 
(white tufts on the surface-sterilised needles) in primary isolations after several 
weeks of cultivation. In pure cultures, this synanamorph also dominated and 
Chalara-like phialides were formed relatively rarely. Penicillate conidiophores 
and phialides were never observed growing from the same hyphae, but potential 
contamination of surveyed strains may be disproved as individual isolates were 
obtained independently from three distant localities. 

It remains unknown whether I. minuta forms predominantly penicillate 
synanamorph also on field substrates. Similarly, Crous & Groenewald (2010) 
do not mention the prevalence of either form of X. pinea on natural substrates. 
The assumption that penicillate conidiophores may dominate on the substrate 
as well is indicated by the white colonies formed by an unknown Polyscytalum- 
like fungus that have been repeatedly found on Scots pine needles sampled 
close to the type locality of I. minuta and cultivated in damp chambers (Koukol 
2007). Herbarium specimen “Polyscytalum sp. X07” (private herbarium of the 
author) sampled in September 2000 and surveyed in this study contained a 
fungus producing relatively dense growth of Polyscytalum-like conidiophores 
(12-18 x 2 um) with chains of cylindrical, 0-1-septate, hyaline conidia (5-13 
x 1.5 um). Except for the septate conidia, its description matches I. minuta, 
but a fresh collection must first be isolated into pure culture and molecularly 
analyzed before it can be regarded as conspecific with I. minuta. 

All I. minuta strains were obtained from pine litter, but the species also 
colonizes Scots pine humus according to 99% similarity (505/507 bp) of the ITS 
rDNA region with sequence GU559086 obtained in the study of Lindahl et al. 
(2010). Infundichalara minuta seems to have a negligible effect on degradation 
of structural polysaccharides in the litter. In a parallel study, strain CCF4156, 


Infundichalara minuta sp. nov.... 351 


analyzed for the production of ten hydrolytic enzymes during submersed 
cultivation in a low-nitrogen medium, expressed only very low amounts of 
cellobiohydrolase and B-glucosidase (Koukol & Baldrian 2012). 


Acknowledgments 

Iam very grateful to Dr. David Svoboda, who sampled litter needles on his journey 
across Russia, to Dr. Pedro Crous for sending me two strains of X. pinea. Dr. Pedro 
Crous and Dr. Roland Kirschner significantly contributed to the improvement of the 
manuscript as pre-submission reviewers. This study was supported by the Grant Agency 
of the Czech Republic, project No. 206/09/P295 and by the Ministry of Education, Youth 
and Sports of the Czech Republic (MSM0021620828). 


Literature cited 

Crous PW, Groenewald JZ. 2010. Fungal planet 55. Xenopolyscytalum pinea. Persoonia 25: 
130-131. 

Crous PW, Schubert K, Braun U, Hoog GS de, Hocking AD, Shin H-D, Groenewald JZ. 2007. 
Opportunistic, human-pathogenic species in the Herpotrichiellaceae are phenotypically similar 
to saprobic or phytopathogenic species in the Venturiaceae. Studies in Mycology 58: 185-217. 

Edgar RC. 2004. MUSCLE: a multiple sequence alignment method with reduced time and space 
complexity. BMC Bioinformatics 5: 113. http://dx.doi.org/10.1186/1471-2105-5-113 

Fassatiova O. 1986. Moulds and filamentous fungi in technical microbiology. Amsterdam, 
Elsevier. 

Gams W, Holubova-Jechova V. 1976. Chloridium and some other dematiaceous hyphomycetes 
growing on decaying wood. Studies in Mycology 13: 1-99. 

Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk W, Gascuel O. 2010. New algorithms and 
methods to estimate maximum-likelihood phylogenies: Assessing the performance of PhyML 
3.0. Systematic Biology 59: 307-321. http://dx.doi.org/10.1093/sysbio/syq010 

Hall TA. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program 
for Windows 95/98/NTT. Nucleic Acids Symposium Series 41: 95-98. 

Holubova-Jechova V. 1978. Lignicolous hyphomycetes from Czechoslovakia 5. Septonema, 
Hormiactella and Lylea. Folia Geobotanica Phytotaxonomica 13: 421-442. 

Kirk PM. 1983. New or interesting microfungi. IX. Dematiaceous hyphomycetes from Esher 
Common. Transactions British Mycological Society 80: 449-467. 
http://dx.doi.org/10.1016/S0007-1536(83)80041-2 

Koukol O. 2007. Effect of Pinus strobus L. invasion on the mycoflora of pine litter needles in the 
Bohemian Switzerland National Park (Czech Republic). 229-233, in: H Hartel et al. (eds). 
Sandstone Landscapes. Academia, Prague. 

Koukol O. 2010. Revision of “Septonema ochraceum” revealed three new species of Venturiaceae 
and Herpotrichiellaceae. Mycological Progress 9: 369-378. 
http://dx.doi.org/10.1007/s11557-009-0645-x 

Koukol O. 2011. New species of Chalara occupying coniferous needles. Fungal Diversity 49: 75-91. 
http://dx.doi.org/10.1007/s13225-011-0092-2 

Koukol O, Baldrian P. 2012. Intergeneric variability in enzyme production of microfungi from pine 
litter. Soil Biology & Biochemistry 49: 1-3. http://dx.doi.org/10.1016/j.soilbio.2012.02.004 

Nag Raj TR, Kendrick WB. 1975. A monograph of Chalara and allied genera. Wilfr. Laurier Univ 
Press, Waterloo 


352 ... Koukol 


Lindahl BD, de Boer W, Finlay RD. 2010. Disruption of root carbon transport into forest humus 
stimulates fungal opportunists at the expense of mycorrhizal fungi. ISME Journal 4: 872-881. 
http://dx.doi.org/10.1038/ismej.2010.19 

Réblova M, Gams W, Stépanek V. 2011. Thenewhyphomycete genera Brachyalara and Infundichalara, 
the similar Exochalara and species of ‘Phialophora sect. Catenulatae’ (Leotiomycetes). Fungal 
Diversity 46: 67-86. http://dx.doi.org/10.1007/s13225-010-0077-6 

Rehner SA, Buckley E. 2005. A Beauveria phylogeny inferred from nuclear ITS and EF1-a sequences: 
evidence for cryptic diversification and links to Cordyceps teleomorphs. Mycologia 97: 84-98. 
http://dx.doi.org/10.3852/mycologia.97.1.84 

Ronquist F, Huelsenbeck JP. 2003. MrBayes 3: Bayesian phylogenetic inference under mixed 
models. Bioinformatics 19: 1572-1574. http://dx.doi.org/10.1093/bioinformatics/btg180 

Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. 2011. MEGAS5: Molecular 
Evolutionary Genetics Analysis using Maximum Likelihood, Evolutionary Distance, and 
Maximum Parsimony Methods. Molecular Biology and Evolution. 
http://dx.doi.org/10.1093/molbev/msr121 


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MY COTAXON 


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Volume 120, pp. 353-355 April-June 2012 


Clavulicium hallenbergii, a new corticioid species 
from India 


AVNEET P. SINGH, JASPREET KAUR & G.S. DHINGRA* 


Department of Botany, Punjabi University 
Patiala 147 002, India 
*CORRESPONDENCE TO: dhingragurpaul@gmail.com 


ABSTRACT - A new corticioid species, Clavulicium hallenbergii, is described from Chamba, 
in Himachal Pradesh. 


Key worps -— Manimahesh, Pinus wallichiana 


During the fungal forays conducted in Dibri-Dhansho area in district Chamba 
of Himachal Pradesh, India, Avneet P. Singh collected a corticioid fungus on a 
trunk of Pinus wallichiana. Oil-rich clavate basidia and oil-rich basidiospores 
pointed in the direction of the genus Clavulicium (Agaricomycetes). After 
detailed comparison of macroscopic and microscopic characters with the 
relevant literature (Burt 1926, Parmasto 1968, Eriksson & Ryvarden 1973) we 
concluded that the fungus represents a new species in the genus Clavulicium, 
which herewith is described. 


Clavulicium hallenbergii Avneet P. Singh, J. Kaur & Dhingra sp. nov. 
MycoBAnk MB 560931 FIGs 1-5 
Differs from Clavulicium macounii by its narrower basidia, smaller basidiospores, and 


lack of gloeocystidia. 


Type: India, Himachal Pradesh: Chamba, Manimahesh, on way from Dibri towards 
Dhansho, on the trunk of Pinus wallichiana A.B. Jacks., 20 September 2005, Avneet 3683 
(PUN, holotype) 


Erymo.ocy: In honour of the mycologist Prof. Nils Hallenberg, University of 
Gothenburg, Gothenburg, Sweden. 
Basidiocarp resupinate, adnate, effused, up to 175 um thick in section; 
hymenial surface smooth, brownish red to light brown; margins whitish, 
thinning, fibrillose, to indeterminate, loosening upon drying in mature 


354 ...Singh, Kaur & Dhingra 


10 poo at 


BOCOO0 


20 ym 


pole 
\ iN a 


Fics 1-4. Clavulicium hallenbergii, microscopic structures. 
1. basidiospores; 2. basidia; 3. generative hyphae; 4. section through basidiocarp. 


basidiocarp. Hyphal system monomitic; generative hyphae up to 3.0 um wide, 
branched, septate, clamped; basal hyphae somewhat thick-walled, parallel to 
the substrate; subhymenial hyphae thin-walled, vertically directed. Basidia 
39.0-47.0 x 3.0-5.0 um, clavate, sinuous, 4-sterigmate, with oily contents and 
basal clamp; sterigmata up to 3.0 um long. Basidiospores 6.2-7.8 x 4.5-5.5 um, 
broadly ellipsoid to obovate, smooth, thin-walled, inamyloid, acyanophilous, 
with oil droplets. 


REMARKS—C. hallenbergii resembles C. macounii (Burt) J. Erikss. & Boidin 
ex Parmasto in terms of morphology of the basidiocarp, clamped generative 


Clavulicium hallenbergii sp. nov. (India) ... 355 


Fic. 5. Clavulicium hallenbergii, basidiocarp (holotype). 


hyphae and abundant oily contents in basidia and basidiospores. However, 
C. macounii differs in having gloeocystidia, wider basidia, and larger 
basidiospores (9-12 (-13) x 6.5-8 um; Eriksson and Ryvarden, 1973). 


Acknowledgements 

Authors thank Prof. Nils Hallenberg (Evolutionary Botany, Botanical Institute, 
Gothenburg, Sweden) and Prof. B.M. Sharma (Department of Plant Pathology, COA, 
CSKHPAU, Palampur, H.P., India) for peer review and Prof. Joost Stalpers (CBS 
Fungal Biodiversity Centre, Netherlands) for providing the Mycobank number. Head, 
Department of Botany, Punjabi University, Patiala, is thanked for providing research 
facilities. 


Literature cited 

Burt EA. 1926. The Thelephoraceae of North America: XV. Corticium and supplement to the whole 
series. Ann. Mo. Bot. Gard. 13: 173-357. 

Parmasto E. 1968. Conspectus systematis Corticiacearum. Tartu. 262 pp. 

Eriksson J, Ryvarden L. 1973. Corticiaceae of North Europe - II. Fungiflora, Oslo. pp 59-286. 


ISSN (print) 0093-4666 © 2012. Mycotaxon, Ltd. ISSN (online) 2154-8889 


MY COTAXON 


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Volume 120, pp. 357-360 April-June 2012 


Vararia longicystidiata sp. nov. (Agaricomycetes) from India 


SAMITA, S.K. SANYAL’, G.S. DHINGRA & AVNEET P. SINGH 


Department of Botany, Punjabi University, Patiala 147 002, India 
*CORRESPONDENCE TO: skskumar731@gmail.com 


ABSTRACT - A new corticioid species, Vararia longicystidiata, is described on decaying wood 
of Quercus incana from Uttarakhand state in India. 


Key worps - Basidiomycota, Chaurangi Khal, Uttarakashi 


While conducting fungal forays in Chaurangi Khal area of district Uttarakashi, 
Uttarakhand (India), Samita collected an unknown corticioid fungus on 
decaying wood of Quercus incana. The presence of dichohyphidia and 
inamyloid smooth basidiospores indicated that the species belonged to Vararia, 
but it could not be assigned to any previously described species (Welden 1965, 
Parmasto 1970, Boidin & Lanquetin 1976, Rattan 1977, Boidin et al. 1980, 
Hallenberg & Eriksson 1985, Pegler & Young 1993). A detailed description of 
the specimen along with the microphotographs and line diagrams was sent 
to Prof. Nils Hallenberg (Sweden), who supported the description of a new 
species in the genus Vararia. 


Vararia longicystidiata Samita, Sanyal, Dhingra & Avneet P. Singh, sp. nov. 
MycoBAnk 562081 Fics 1-7 
Differs from Vararia gomezii in its larger cystidia, basidia, and basidiospores. 
Type: India, Uttarakhand: Uttarakashi, Chaurangi Khal, on decaying wood of Quercus 
incana Bartram, 29 September 2011, Samita 4413 (PUN, holotype). 


Erymo.oey: The epithet refers to the long cylindrical cystidia. 


Basidiocarp resupinate, adnate, effused, up to 230 um thick in section, 
hymenial surface smooth to somewhat tuberculate, light orange; margins 
thinning, fibrillose, paler concolorous to whitish. Hyphal system monomitic; 
generative hyphae up to 5.5 um wide, branched, septate, generally without 
clamps; basal hyphae loosely arranged, more or less parallel to the substrate, 
interspersed with some thick-walled hyphae with oily contents, some cells 


358 ... Samita & al. 


10nm 


— 
- 
- 
-— 


~——_ ae 


microscopic structures. 


Fics 1-6. Vararia longicystidiata, 


1. Basidiospores; 2. basidia; 3. generative hyphae; 4. cystidia; 


5. dichohyphidia; 6.section through basidiocarp. 


Vararia longicystidiata sp. nov. (India) ... 359 


Fic. 7. Vararia longicystidiata, basidiocarp (holotype). 


inflated; subhymenial hyphae vertical, compactly arranged. Dichohyphidia 
more common in subhymenium than hymenium, abundant, coralloid, 
branches irregular with blunt endings, dextrinoid. Cystidia 100.0-188.0 x 
8.8-17.5 um, abundant, subcylindrical to sinuous, thick-walled, without basal 
clamp, negative to sulphovanillin. Basidia 70.0-110.0 x 9.0-13.0 um, clavate, 
4-sterigmate, frequently with secondary septa, without basal clamp, with or 
without oily contents; sterigmata up to 12.5 um long; basidioles rich in oily 
contents. Basidiospores 15.0-20.0 x 6.0-7.5 um, sub-fusiform to navicular, 
with numerous oil-drops, thin- to somewhat thick-walled, inamyloid, 
acyanophilous. 


REMARKS—Vararia longicystidiata resembles V. gomezii Boidin. & Lanq. 
(reported from South America and Africa) in having similar basidiocarps and 
basidiospore shape. However, V. gomezii differs in having ovoid unthickened 
cystidia (14-50 x 8-14 um), smaller basidia (34.0-45.0- x 7.5), and smaller 
basidiospores (13.0-14.8 x 4.8-6. 25 um; Boidin and Lanquetin; 1977). 


Acknowledgements 
The authors thank Head, Department of Botany, Punjabi University, Patiala, 
for providing research facilities; Prof. Nils Hallenberg (University of Gothenburg, 


360 ... Samita & al. 


Gothenburg, Sweden) for expert comments and peer review; Prof. B.M. Sharma 
(Department of Plant Pathology, COA, CSKHPAU, Palampur, H.P., India) for peer 
review. 


Literature cited 

Boidin J, Lanquetin P. 1976 [“1975”]. Vararia subgenus Vararia (Basidiomycetes Lachnocladiaceae): 
étude spéciale des espéces d'Afrique intertropicale. Bull. Soc. Mycol. France 91(4): 457-513. 

Boidin J, Lanquetin P. 1977. Les genres Dichostereum et Vararia en Guadeloupe (Basidiomycetes, 
Lachnocladiaceae). Mycotaxon 6: 277-336 

Boidin J, Lanquetin P, Gilles G. 1980. Application du concept biologique de Tespéces aux 
basidiomycetes: le genre Vararia (section Vararia) au Gabon. Cryptog. Mycol. 1: 265-384. 

Hallenberg N, Eriksson J. 1985. The Lachnocladiaceae and Coniophoraceae of North Europe. 
Fungiflora, Oslo. pp. 1-96. 

Pegler DN, Young TWK. 1993. Basidiome structure in Lachnodiales sensu lato. Kew Bullein 48: 
oye. 

Parmasto E. 1970. The Lachnocladiaceae of the Soviet Union with a key to boreal species. Scripta 
Mycologia 2: 1-168 

Rattan SS. 1977. The resupinate Aphyllophorales of the northwestern Himalayas. Bibliotheca 
Mycologica 60: 1-427. 

Welden AL. 1965. West Indian species of Vararia with notes on extralimital species. Mycologia 57: 
502-520. 


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MY COTAXON 


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Volume 120, pp. 361-371 April-June 2012 


A new cystidiate variety of Omphalina pyxidata (Basidiomycota, 
tricholomatoid clade) from Italy 


ALFREDO VIZZINI’*, MARIANO CurRTI’?,, MARCO CONTU? & ENRICO ERCOLE! 


‘Dipartimento di Scienze della Vita e Biologia dei Sistemi - Universita degli Studi di Torino 
Viale Mattioli 25, I-10125, Torino, Italy 

? Via Tito Nicolini 12, Pozzaglia Sabina, I-02030 Rieti, Italy 

> Via Marmilla 12 (I Gioielli 2), I-07026 Olbia (OT), Italy 


*CORRESPONDENCE TO: alfredo. vizzini@unito. it 


ABSTRACT — A new variety of Omphalina pyxidata, var. cystidiata, is here described and 
illustrated based on morphological and molecular data. The new combination Infundibulicybe 
lateritia is introduced. 


Key worps — Agaricomycetes, Agaricales, omphalinoid fungi, Contumyces, taxonomy 


Introduction 

Within the omphalinoid fungi — small agarics with a convex to deeply 
umbilicate pileus, central stipe, thin context, decurrent lamellae, white spore- 
print, and thin-walled inamyloid smooth spores (Norvell et al. 1994, Lutzoni 
1997, Redhead et al. 2002a,b) — taxa with cystidiate basidiomata are thus far 
known only in the hymenochaetoid clade (Blasiphalia Redhead, Contumyces 
Redhead et al., Rickenella Raithelh.; Moncalvo et al. 2000, 2002, Redhead et al. 
2002b, Larsson et al. 2006, Larsson 2007). 

During fieldwork focused on bryophilous Galerina species, we collected an 
omphalinoid fungus growing on mosses close to Galerina discreta E. Horak 
et al. We initially believed that these specimens represented a new species 
of Contumyces (Contu 1997, Redhead et al. 2002b) based on the presence of 
well-differentiated pileo-, caulo-, cheilo-, and pleurocystidia and an irregular 
hymenophoral trama. However an ITS-rDNA analysis implied they might 
represent an unknown as-yet undescribed variety of Omphalina pyxidata (Bull.) 
Quél., the type species of Omphalina Quél. (Omphalina s.s. = tricholomatoid 
clade sensu Matheny et al. 2006, Binder et al. 2010 and Vizzini et al. 201 1a). This 
genus is characterized by bryophilous basidiomata with a reddish brown tinged 


362 ... Vizzini & al. 


pileus and stipe, paler and well-developed lamellae, a smooth (not scaly) pileus, 
absence of hymenial and pileal cystidia, and presence of clamp-connections 
(Redhead et al. 2002a, Elborne 2008). We fully describe and illustrate the new 
taxon below. 


Materials & methods 


Morphology 

The macromorphological descriptions follow the detailed field notes taken on fresh 
material. The micromorphological descriptions are based both upon study of fresh 
and herbarium material. Dried material was revived in 5% KOH and stained in Congo 
Red, Cotton Blue, and Melzer’s reagent. The basidiospore range and means are based 
on measurements of 25 spores. In the macro- and microscopic descriptions Q = the 
quotient of spore length and width and Qm = the average quotient; L = number of entire 
lamellae; | = number of lamellulae between each pair of entire lamellae. The basidial 
width was measured at the broadest part, and the length was measured from the apex 
(sterigmata excluded) to the basal septum. Colour codes in brackets (e.g., Se 45) follow 
Séguy (1936), hereafter referred to as (Se). Author citations follow the IPNI Authors 
and the Index Fungorum Authors of Fungal Names websites. Herbarium abbreviations 
follow Thiers (2011) with the exception of “EM” and “GT”, referring to the personal 
herbaria of Enzo Musumeci and Gérard Trichies, respectively. The type collection is 
housed at TO (Herbarium of the Department of Plant Biology, University of Turin, Italy), 
and both name and Latin diagnosis of the new variety as well as the new combination 
are deposited in MycoBank (http://www.mycobank.org). 


DNA extraction, PCR amplification, DNA sequencing 

Genomic DNA was isolated from 1 mg of 3 herbarium specimens (TABLE 1) using the 
DNeasy Plant Mini Kit (Qiagen, Milan Italy). Universal primers ITS1£/ITS4 were used 
for the ITS region amplification (White et al. 1990; Gardes & Bruns 1993). Amplification 
reactions were performed in PE9700 thermal cycler (Perkin-Elmer, Applied Biosystems) 
in a 25 ul reaction mixture using the following final concentrations or total amounts: 
5 ng DNA, 1x PCR buffer (20 mM Tris/HCl pH 8.4, 50 mM KCl), 1 uM of each primer, 
2.5 mM MgCl2, 0.25 mM of each dNTP, 0.5 unit of Taq polymerase (Promega). The 
PCR program was as follows: 3 min at 95°C for 1 cycle; 30 s at 94°C, 45 s at 50°C, 
2 min at 72°C for 35 cycles, 10 min at 72°C for 1 cycle. PCR products were resolved on a 
1.0% agarose gel and visualized by staining with ethidium bromide. PCR products were 
purified and sequenced by DiNAMYCODE srl (Turin, Italy). Sequence assembly and 
editing were performed using Geneious v5.3 (Drummond et al. 2010). The sequences 
are deposited in GenBank under the accession numbers given in TABLE 1. 


Sequence alignment and phylogenetic analysis 

Sequences included in the phylogenetic analyses were either generated in this study 
(TABLE 1) or retrieved from GenBank. Multiple sequence alignments for ITS fragments 
were generated using MAFFT (Katoh et al., 2002) with default conditions for gap opening 
and gap extension penalty. The alignment was slightly edited using MEGA 5.0 (Tamura 
et al. 2011). Phylogenetic analyses were performed using both Bayesian Inference (BI) 


Omphalina pyxidata var. cystidiata var. nov. (Italy) ... 363 


TABLE 1. Omphalina pyxidata collections used in this study. 


COLLECTIONS ! COLL. NO., COUNTRY, DATE, COLLECTOR ! ITS Acc. No. 


. pyxidata , France, 12/1 , G. Trichies 1 
*O. pyxidata 1 GT99398, F 02/12/1999, G. Trich JQ671000 


*O. pyxidata var. cystidiata : TO HG2512, Italy, 11/11/2010, M. Curti : JQ671002 


* = collections newly sequenced in this study. 


and Maximum Likelihood (ML) approaches. The BI was performed with MrBayes 
3.1.2 (Huelsenbeck & Ronquist 2001) with four incrementally heated simultaneous 
Monte Carlo Markov Chains (MCMC) ran over 10 millions generations, under GTR+I 
evolutionary model. Trees were sampled every 1000 generations resulting in an overall 
sampling of 10,001 trees; the first 2500 trees were discarded as “burn-in” (25%). For the 
remaining trees, a majority rule consensus tree showing all compatible partitions was 
computed to obtain estimates for Bayesian Posterior Probabilities (BPP). Branch lengths 
were estimated as mean values over the sampled trees. ML was performed with RAXML 
(Stamatakis 2006) under GTRGAMMA model and using thorough bootstrap with 
20 runs and 1000 replicates. In both analyses a Pseudoclitocybe cyathiformis sequence 
(Genbank HM191730) was used as outgroup taxon, according to Binder et al. (2010) 
and Vizzini et al. (2011a). The ML consensus tree was used merely for comparison with 
the Bayesian tree and to support the analysis. However the BPP (Bayesian posterior 
probability) over 0.75 and the ML bootstrap (MLB) over 50% values are reported in the 
resulting tree. Pairwise % identity values of ITS sequences were calculated using MEGA 
5.0 (Tamura et al. 2011). 


Results 


Molecular results 

Bayesian and Maximum likelihood inferences were performed on a total 
of 18 samples, including 15 sequences available from GenBank and three 
newly sequenced specimens (TABLE 1). Final alignment length was 731 bp. 
Both Bayesian and Maximum likelihood analyses produced the same topology 
(Fic. 1). In both analyses our cystidiate collection clusters with four O. pyxidata 
sequences clearly forming a strongly supported clade (BPP 0.92 and MLB 85%). 
The five O. pyxidata sequences show a pairwise % identity value of 99.8 and 
could be considered conspecific. 

The O. pyxidata sequence deposited in Genbank under the accession number 
JF908502 (MCVE 15669) was not used in the analysis because a Blastn search 


364 ... Vizzini & al. 


Omphalina pyxidata 3 JN944402 


0.77/90 
Omphalina pyxidata 4 OPU66450 


Omphalina pyxidata 1 JQ671000 
0.92/85 
Omphalina pyxidata var. cystidiata JQ671002 

Ompbhalina pyxidata 2 JQ671001 

Omphalina rivulicola GU234117 
diss Omphalina cf. rivulicola HQ445617 
Omphalina rivulicola ORU66451 

Omphalina chionophila GU234144 

Omphalina rivulicola JF908501 

Clitocybe lateritia CLU66431 
Omphalina cf. rivulicola HQ445603 
0.76/84 Omphalina rivulicola GU234109 
Infundibulicybe gibba DQ490635 
Infundibulicybe gibba HM631716 
Infundibulicybe mediterranea HM631724 


Clitocybe alkaliviolascens JF907807 


Pseudoclitocybe cyathiformis HM191730 


0.03 expected changes per site 


FicuRe 1. Bayesian phylogram obtained from the ITS sequence alignment. Support values for clades 
that are supported in either the Bayesian (Posterior Probabilities values - BPP) and Maximum 
likelihood (ML Bootstrap percentage - MLB) analyses are indicated. BPP above 0.75 and MLB 
above 50% are given above branches. Taxa in bold represent newly sequenced collections. Numbers 
(1-4) refer to the collections reported in TABLE I. 


implies it represents a non-omphalinoid species in the Pluteus cinereofuscus/ 
P. nanus complex. 

Clitocybe lateritia, together with two O. rivulicola (J. Favre) Lamoure 
collections, clearly falls within Infundibulicybe. Clitocybe alkaliviolascens Bellu 
clusters close to Infundibulicybe mediterranea Vizzini et al. 


Taxonomy 


Omphalina pyxidata var. cystidiata M. Curti, Contu & Vizzini, var. nov. Fics 2-3 
MycoBank MB564484 


A varietate typica differt praesentia cystidiorum in lamella, pileo, et stipite. Habitat: 
graegatim, ad muscos. 


Type — Italy, Latium, prov. Rieti, com. Pozzaglia Sabina, Valle del Turano, 42.1596°N 
12.9652°E, 878 m, 11.X1.2010, leg. M. Curti (TO HG2512 holotype). 


EryMoLocy — the specific epithet refers to the presence of hymenial as well as of pileo- 
and caulocystidia. 


Omphalina pyxidata var. cystidiata var. nov. (Italy) ... 365 


FiGuRE 2. Omphalina pyxidata var. cystidiata. Basidiomata. Bar = 20 mm. 


Piteus 3-15 mm broad, at first convex to applanate, later with a slightly 
depressed centre, not or slightly hygrophanous, minutely pubescent, dark 
brown-fulvous (Se 96, 121, 126, 146, 251, 252) fading to light brown (Se 133, 
134, 173); margin at first involute then expanding, smooth to slightly striate in 
old basidiomes, whitish in dried basidiomes. LAMELLAE (L = 18-25; ] = 1-2) 
decurrent, thick, distant with lamellulae occasionally interspersed, strongly 
forked and intervenose, whitish to light ochre (Se 199-200), with entire 
concolorous edge. Stripe 15-40 x 1-3 mm, cylindrical, slightly broadened at 
the base, sinuous-flexuous, solid then fistulous, cartilaginous, paler than pileus, 
light ochre-brown (Se 133, 174, 203-204), minutely pruinose to subglabrous. 
CoNnTEXT elastic, thin, whitish-ochre to light ochre-brown (Se 133, 134) in 
surface; smell and taste not distinctive. SPORE PRINT white. 

SPORES (7-)7.5-9(-10) x 4.5-6(-6.5) um, Q = 1.3-1.7, Qm = 1.51, largely 
ellipsoid to oblong in frontal view, lacrymoid to slightly amygdaliform in side- 
view, smooth, thin-walled, hyaline, inamyloid, mono-biguttulate, with a thick 
and evident hilum. Basrp1a 28-46(-60) x 6-9 um, clavate, (two-) four-spored, 
sterigmata 3-7 um long, usually clamped. CHEILOCysTIDIA 23-45 x 3-6 x 
2.5-5 um, abundant, cylindrical, slender, apically tapered or slightly swollen, 
some laterally ventricose, hyaline, thin-walled, often with inner microguttules. 
PLEUROCYSTIDIA 25-50(-65) x 3.5-5.5 x 2.5-5(—7) um, similar to cheilocystidia, 
but slightly longer. HYMENOPHORAL TRAMA irregular, consisting of 3.3-7 um 
wide hyphae. PILEIPELLIS a cutis consisting of cylindrical, interwoven, 6-10 


366 ... Vizzini & al. 


FIGURE 3. Omphalina pyxidata var. cystidiata. Microscopic features. a. Spores showing protruding 
hilum. b. Basidium with basal clamp. c. Pileipellis and pileocystidia. d. Encrusting pigment of 
pileipellis elements. e. Cheilocystidia (with inner microguttules). f. Pleurocystidia. g. Caulocystidia. 
h. Irregular hymenophoral trama. Bars: a~d = 10 um; e—h = 20 um. 


Omphalina pyxidata var. cystidiata var. nov. (Italy) ... 367 


um wide hyphae; terminal elements with a subclavate 7-13 um wide apex; 
subcutis made up of 3-7 um wide hyphae; PIGMENT epiparietal, minutely to 
strongly encrusting; PILEOCYSTIDIA numerous, fusiform to lageniform, 35-60 
x 3.5-9 x 3.5-5 um. STIPITIPELLIS a cutis made up, in 5% KOH, of ochre-yellow 
3-8 um wide hyphae bearing minute incrustations; CAULOCYSTIDIA similar to 
hymenial cystidia (30-65 x 4-5 x 3-5 um). STIPITITRAMA non-sarcodimitic, 
consisting of hyphae up to 16 um wide. CLAMP CONNECTIONS present at all 
septa. THROMBOPLEROUS HYPHAE (= oOleiferous hyphae sensu Clémencon 
2004) present. APPRESSORIA on host surface not observed. 
HaBItatT — Gregarious, rarely subcaespitose, on Tortula muralis 

gametophytes (Bryophyta). 

ADDITIONAL MATERIAL STUDIED — Omphalina pyxidata var. pyxidata: FRANCE, 

LORRAINE, MOSELLE, Moyeuvre-Petite, 02 Dec 1999, on mosses, leg. G. Trichies 

(GT99398); SWITZERLAND, CANTON BasEL StTapT, Lange Erlen, 06 Jul 2005, 

on mosses, 250 m asl, leg. E. Musumeci (EM0434-05); ITALY, PrEDMONT, CHISONE 


VALLEY, Pinerolo, Pra Martino, 10 Oct 2010, on mosses, 1000 m asl, leg. A. Vizzini (TO 
AV98). 


Discussion 

As highlighted by Vizzini et al. (201la), Omphalina s.s. is sister to 
Infundibulicybe Harmaja, a genus recently segregated from Clitocybe (Fr.) 
Staude (Harmaja 2003). In the six-gene region sequence analyses by Matheny & 
al. (2006), Infundibulicybe falls outside the tricholomatoid clade, occupying an 
isolated position in the Agaricales (incertae sedis). However, recent analyses by 
Binder et al. (2010), Vizzini et al. (2011a) and Matheny (pers. comm.) place it 
with robust support as sister to the rest of the tricholomatoid clade. Omphalina 
and Infundibulicybe share the cream-reddish brown tinges of pileus and stipe, 
the long-decurrent lamellae, and strongly encrusting pigment (Harmaja 2003, 
Vizzini et al. 2011b). 

Our molecular analysis (Fic. 1) and morphological comparison support 
the new taxon as a cystidiate variant of O. pyxidata thus far never described. 
Omphalina pyxidata var. cystidiata is unique within Omphalina s.s., where it is 
the only taxon thus far included that possesses well-developed cheilo-, pleuro- 
, pileo-, and caulocystidia (Bigelow 1970, 1985, Singer 1970, Lamoure 1974, 
1975 1982; Clémencon 1982, Bon 1997, Elborne 2008). Furthermore, we have 
found no cystidia in three examined European collections of O. pyxidata (see 
ADDITIONAL MATERIAL STUDIED). Morphologically, our new taxon resembles 
the omphalinoid bryophilous genera belonging to the hymenochaetoid clade 
(Redhead et al. 2002b, Antonin & Noordeloos 2004). However, Rickenella 
differs in having a regular hymenophoral trama (Contu 1997, Redhead et al. 
2002b) and Blasiphalia by growing specifically on the Blasia pusilla gametophyte 
(Marchantiophyta) and forming appressoria on host surface (Larsson et al. 


368 ... Vizzini & al. 


2006). Apart from molecular data that clearly support our new variety within 
the Agaricales, morphologically Contumyces (= Jacobia Contu, nom. illeg.; 
Hymenochaetales) might accommodate our taxon. Contumyces so far comprises 
only three species — C. rosellus (M.M. Moser) Redhead et al., C. vesuvianus 
(F. Brig.) Redhead et al., and C. brunneolilacinus (Contu et al.) Redhead et al. 
(Redhead et al. 2002b, Antonin & Noordeloos 2004) — but only the first named 
has been sequenced. Contumyces rosellus differs from Omphalina pyxidata var. 
cystidiata by the pink to pale salmon-pink colouration, white stipe, and longer 
and larger cystidia. Contumyces vesuvianus has orange to fulvous basidiomata, 
larger basidiospores, rarer differently shaped (mucronate/rostrate) cystidia 
and intracellular pigment in the pileipellis hyphae. As originally described 
(Contu 1989), C. brunneolilacinus differs in having a lilaceous-brown, sharply 
tomentose, hygrophanous pileus, pink-lilac lamellae, a Pelargonium-like smell, 
lageno-capitate cystidia, and intracellular pigment in the pileipellis hyphae. 
Finally, C. brunneolilacinus sensu Antonin & Noordeloos (2004) differs in 
having longer and larger cystidia and a non-tomentose pileal surface. 

Our sequence analyses have also revealed that Clitocybe lateritia clusters 
in Infundibulicybe with two collections clearly misdetermined as O. rivulicola 
(Fic. 1). The three sequences are clearly conspecific (pairwise % identity 
value = 99.7). The two “O. rivulicola” sequences were derived from soil or 
ectomycorrhizal samples sampled from Dryas octopetala beds, as reported in two 
recent papers dealing with alpine-arctic fungi (Bjorbeekmo et al. 2010, Geml et 
al. 2012). Clitocybe lateritia, a rare alpine-arctic species strictly associated with 
Dryas octopetala (Rosaceae), should be transferred to Infundibulicybe based on 
both sequence analyses and its Infundibulicybe-like features (reddish-brown 
colouration, strongly encrusting pigment in the pileipellis, partly lacrymoid 
spores) as inferred from Favre (1955), Gulden & Jennsen (1988), and Bon 
(1997). Consequently, we propose the following new combination: 


Infundibulicybe lateritia (J. Favre) Vizzini & Contu, comb. nov. 
MycoBank MB564485 
= Clitocybe lateritia J. Favre, Ergebn. wiss. Unters. schweiz. 
Natn. Parks, n.s. 5(33): 54, 199 (1955). 


Type: Switzerland, Munt la Schera, 2400 m a.s.l., in Dryas beds, 21.08.1941, leg. J. Favre 
(Lectotype designated here, G-K16433; specimen illustrated in Favre 1955: Fig. 34; Pl. 
IV, Fig. 11). 


Finally, Clitocybe alkaliviolascens, a species of the Infundibulicybe gibba complex 
recently described from Mediterranean areas and characterized by a dark brown 
pileus surface turning pink-violet with a 30% KOH drop, and spores reaching 
10 um in length (Bellu 1995, 1996), also belongs in Infundibulicybe. Bellu will 
propose the new combination in a forthcoming paper (Bellu, pers. comm.). 


Omphalina pyxidata var. cystidiata var. nov. (Italy) ... 369 


Acknowledgements 

Our most sincere thanks are due to Scott Redhead (Ottawa, Ontario, Canada), Lorelei 
Norvell (Portland, Oregon, USA), and Shaun Pennycook (Auckland, New Zealand) for 
their pre-submission reviews. We would like also to thank Luca Miserere (Torino, Italy) 
for identifying the moss species and Enzo Musumeci (Basel, Switzerland), Philippe 
Clerc (Chambésy, Switzerland) and Gérard Trichies (Neufchef, France) for sending 
herbarium specimens. 


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ISSN (print) 0093-4666 © 2012. Mycotaxon, Ltd. ISSN (online) 2154-8889 


MY COTAXON 


http://dx.doi.org/10.5248/120.373 
Volume 120, pp. 373-384 April-June 2012 


Four new records of Aspergillus sect. Usti 
from Shandong Province, China 


LonG WANG * 


State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 
100101, China 
* CORRESPONDENCE TO: wl_dgk@yahoo.com.cn 


ABSTRACT—Four new Chinese records of Aspergillus species: Emericella heterothallica, 
Aspergillus calidoustus, A. keveii, and A. pseudodeflectus are reported from Shandong 
Province. Emericella heterothallica, despite being uncommonly reported, should be regarded 
as geographically widely distributed. 


Key worps — morphology, sequence analysis, taxonomy 


Introduction 

Raper & Fennell (1965) recognized five species in the Aspergillus ustus 
group for which Gams et al. (1985) later established subgen. Nidulantes sect. 
Usti. Kozakiewicz (1989) added two species to this section and removed one 
species, while Klich (1993) transferred A. granulosus Raper & Thom from sect. 
Usti to sect. Versicolores. The phenotypic evidence spawned different opinions 
on phylogenetic relationships. 

Peterson (2000) rejected sect. Usti based on Lsu rDNA sequence data (with 
low bootstrap support) and placed the species in sect. Nidulantes. Houbraken 
et al. (2007), who revived A. insuetus (Bainier) Thom & Church based on 
multilocus DNA data along with chemical and phenotypic evidence, accepted 
eight species in sect. Usti; Peterson (2008), accepting the species Houbraken et 
al. included, added seven additional species to the section. Recently, Samson 
et al. (2011) added five new taxa to this section, enlarging to twenty-one but 
without including A. ochraceoroseus and A. versicolor. 

Only three species representing sect. Usti have thus far been recorded in 
China: A. deflectus Fennell & Raper, A. puniceus Kwon-Chung & Fennell, and 
A. ustus (Qi et al. 1997). In the present paper, we report the recovery of four 
additional members of this section: E. heterothallica, A. calidoustus, A. keveii, 


374 ... Wang 


TABLE 1. Strains included in molecular phylogenetic analyses. 


GENBANK ACCESSION NUMBERS “ 


SPECIES STRAINS * 
ITS1-5.8S-ITS2 BenA CaM 
Aspergillus amylovorus Panas. ex Samson NRRL5813 T EF652503 EF652327 EF652415 
A. calidoustus CBS 114380 EF591741 EF591729  EF591716 
CBS 113228 EF591739 EF591730 EF591715 
AS3.15302 JN982696 JN982686 JN982676 
A. deflectus NRRL2206 T EF652437 EF652261 EF652349 
A. egyptiacus Moub. & Mustafa NRRL5920 T EF652504 EF652328 EF652416 
A. elongatus J.N. Rai & S.C. Agarwal NRRL5176 T EF652502 EF652326 EF652414 
A. granulosus NRRL 1932 T EF652430 EF652254 EF652342 
A. insuetus CBS 107.25 T EU076356 EU076371 EU076366 
CBS 119.27 EU076355 EU076372 EU076367 
A. keveii NRRL 1974 EF652432 EF652256 EF652344 
CBS 561.65 EU076352 EU076375 EU076364 
CBS 209.92 T EU076354 EU076376 EU076365 
AS3.15305 JN982704 JN982694 JN982684 
A. lucknowensis J.N. Rai et al. NRRL 3491 T EF652459 EF652283 EF652371 
A. minutus Abbott NRRL 4876 T EF652481 EF652305 EF652393 
NRRL 279 EF652457 EF652281 EF652369 
A. pseudodeflectus NRRL6135 T EF652507 EF652331 EF652419 
NRRL278 EF652456 EF652280 EF652368 
AS3.15306 JN982697 JN982687 JN982677 
AS3.15307 JN982700 JN982690 JN982680 
AS3.15308 JN982699 JN982689 JN982679 
AS3.15309 JN982701 JN982691 JN982681 
AS3.15310 JN982703 JN982693 JN982683 
A. puniceus NRRL 5077 T EF652498 EF652322 EF652410 
A. ustus NRRL 4991 EF652492 EF652316 EF652404 
NRRL 275 T EF652455 EF652279 EF652367 
AS3.15311 JN982695 JN982685 JN982675 
AS3.15312 JN982702 JN982692 JN982682 
A. versicolor (Vuill.) Tirab. NRRL238 T EF652442 EF652266 EF652354 
Emericella heterothallica NRRL 5096 T EF652449 EF652323 EF652411 
NRRL 5097 EF652500 EF652324 EF652412 
AS3.15313 JN982698 JN982688 JN982678 


* Ex-type strains are indicated with “T”. 


A Sequences JN982675-JN982704 were obtained in the present study. 


and A. pseudodeflectus. All were isolated from soil samples collected at the foot 
of Mount Tai in Shandong Province, located in the monsoon area of moderate- 
temperate zone of China (36°15'17"N 117°06'15"E) in late July 2011. The 
average altitude of that area is 134 m with an atmospheric pressure of 1004.1 


Aspergillus spp. new to China... 375 


hPa; the annual average temperature is 12.8 °C, with the monthly average of 
—1.4 °C in January and 26.5 °C in July; the soil is typically frozen from late 
October to late March; the frost-free period is about 200 days from late March 
to late September; the annual precipitation is 700 mm (http://www.weather. 
com.cn). 


Materials & methods 

Soil samples were collected underneath leaf litter and kept in sterilized plastic bags. 
The fungi were isolated through dilution plating (Malloch 1981) using Dichloran Rose 
Bengal Chlortetracycline agar (King et al. 1979) as the selective medium. Ten sect. Usti 
strains were obtained and deposited in the China General Microbiological Culture 
Collection Center, Institute of Microbiology, Chinese Academy of Sciences, Beijing 
(CGMCC). 

Morphological characters were assessed according to Houbraken et al. (2007), Klich 
(2002), and Raper & Fennell (1965). Color names (shown here in title case) follow 
Ridgway (1912). Wet mounts were prepared using material from colonies grown on 
Czapek yeast autolysate agar (CYA) at 25 °C after 14 days mounted in lactophenol 
without dye (Raper & Fennell 1965). Optical microscopic examination and photography 
were performed with a Nikon Eclipse 80i and Nikon Digital Sight DS-L1 Microscope 
(Nikon Co. Ltd., Japan). 

Total DNA extraction followed the method of Scott et al. (2000). For amplification 
of a portion of the B-tubulin gene, we used the primers described by Glass & Donaldson 
(1995); to amplify the ITS1-5.8S-ITS2 region of the Nuc rRNA gene, we used the primers 
designed by White et al. (1990). To amplify a portion of the calmodulin gene, we used 
the following primers: cmdAD1 5'-Gcc GAC TCT TTG ACT GAA GAG C-3’, cmd AD2: 5'- 
GCC GAT TCT TTG ACC GAG GAA C-3' and cmdAD3: 5’-GCc GAT TCT TTG ACC GAA GAA 
c-3' (sense primers); cmdQ1: 5'-GCA TCA TGA GCT GGA CGA ACT C-3’ and cmdQ2?: 5’- 
GCA TCA TGA GCT GGA CGA ATT C-3’ (antisense primers). Polymerase chain reaction 
(PCR) protocols for amplification of the above three gene regions, and the purification 
and sequencing of PCR products followed the methods of Wang & Zhuang (2007). 

Raw sequences were assembled and edited manually with BioEdit 5.0.9 (Hall 
1999). Edited sequences were aligned using Clustal X 1.81 (Thompson et al. 1997), and 
adjusted manually, as needed. Thirty-three strains of sect. Usti (TABLE 1) were analyzed 
using the neighbor-joining (NJ) method and subjected to 1000 bootstrap replicates. All 
the phylograms yielded the same results (e.g., Fic. 5, based on partial calmodulin gene 
sequences). 


Taxonomy 


Emericella heterothallica (Kwon-Chung, Fennell & Raper) Malloch & Cain PL. 1 

Colonies on Czapek agar (CA) at 25 °C growing rapidly, Colonies in 14 
days reaching 40-43 mm in diam., densely floccose to velvety, radially sulcate, 
margins entire; Hiille cells aggregated into small white masses visible to the 
unaided eye, distributed sparsely on the colony surfaces; conidiogenesis sparse, 
only present in the adjacent areas of colonies, brown in color, similar to Verona 


376 ... Wang 


PratE 1. Emericella heterothallica (AS3.15313): A-B. colonies on CA, CYA at 25 °C after 14 days; 
C-D. conidiophores; E. conidia; F. hiille cells. Bar = 10 um 


Brown, mycelia Orange-Cinnamon to Vinaceous-Cinnamon, white at margins; 
exudate abundant, Brazil Red to Strawberry Pink; soluble pigment abundant, 
Congo Pink; reverse Indian Red. 


Aspergillus spp. new to China... 377 


Colonies on CYA at 25 °C growing very rapidly, attaining 50-53 mm in 
diam. in 14 days; texture velvet, radially sulcate with lightly discernable annular 
plications, margins entire; Hiille cells moderately abundant, aggregated 
into conspicuous, small white masses, distributed sparsely on the surfaces; 
conidiogenesis moderate, in marginal areas, Verona Brown; mycelia Cinnamon 
to Vinaceous-Cinnamon, white at colony peripheries; exudate light yellow 
brown, moderately abundant; soluble pigment Empire Yellow, moderately 
abundant; reverse Mars Orange to Orange Rufous. 

Conidial heads globose when young, splitting at maturity into 2-3 loosely 
divergent, short columns, (100-) 120-180 um long; conidiophores arising from 
substratum or surface hyphae, stipes colorless becoming light brown in upper 
portion, thin-walled, 240-360(-420) x (5-)7-9(-11) um; vesicles subglobose 
to ellipsoidal, thin-walled, 14-18(-—22) x 11-14 um, biseriate; metulae 5-6.5 x 
2-3 um; phialides ampuliform, 6.5-7 um, with short collula; conidia globose 
(3-)3.5-5.5 um, echinulate, brown-colored en masse; Hiille cells ellipsoidal to 
very elongate, or joined into botuliform chains, thick-walled, commonly 36-45 
x 9-16 um, smaller ones 16-27 um, longer ones up to 54-63 um. 

Colonies on CYA with 20% sucrose (CY20S) at 25 °C growing very rapidly, 
reaching 52-55 mm in diam. in 14 days, densely floccose to velvety, radially 
sulcate with annular plications, margins entire; conidiogenesis absent; mycelia 
Ochraceous-Buff, white at margins; exudate and soluble pigment absent; 
reverse Nopal Red. 

Colonies on CYA at 37 °C growing moderately, in 14 days attaining 36-38 
mm diam.; texture velvety, flat, margins entire; conidiogenesis moderately 
heavy in central areas, brown-colored near Bister, with a Mikado tinge; mycelia 
white at margins to Baryta Yellow in submarginal areas; exudate and soluble 
pigment absent; reverse Lemon Chrome, with Buff Yellow tinge in centers. 


ISOLATE EXAMINED: China, Shandong Province, foot of Mount Tai, from soil, 23 July 
2011, Z. Yang (CGMCC AS3.15313). 


Aspergillus calidoustus Varga, Houbraken & Samson PL. 2 

Colonies on CA at 25 °C growing moderately, colonies attaining 35-40 mm 
in diam. in 14 days, flat, sparsely floccose, margins fimbriate; conidiogenesis 
sparse, Light Drab; mycelia white; exudate and soluble pigment absent; reverse 
white. 

Colonies on CYA at 25 °C growing rapidly, attaining 48-49 mm in diam. in 
14 days, moderately deep, margins entire, floccose; conidiogenesis heavy, Drab 
Gray to Light Drab; mycelia white; exudate and soluble pigment absent; reverse 
Pale Yellow—Green to Light Viridine Green. 

Conidial heads small, radiate; conidiophores borne from aerial hyphae, 
stipes straight, with light yellow tinge, smooth-walled, (130—)150-250(-300) 


378 ... Wang 


Pl 


Aa 


~~ 


PLATE 2. Aspergillus calidoustus (AS3.15302): A—B. colonies on CYA, CY20S at 25 °C after 14 days; 
C-D. conidiophores; E. conidia. Bar = 10 um 


x 5-7 um; vesicles subglobose, light brown, 9-18(-24) um, biseriate; metulae 
very short, 3.5 x 2-3 um; phialides ampuliform, 7-7.5 x 2-3 um, with short 
collula; conidia spheroidal, conspicuously echinulate, 2.5-3.6(-4) um; Hiille 
cells not found. 

Colonies on CY208S at 25 °C growing rapidly, attaining 46-49 mm in diam. 
in 14 days, moderately deep, depressed at centers, margins entire, floccose; 
conidiogenesis heavy, Drab Gray to Light Drab; mycelia white; exudate and 
soluble pigment absent; reverse Pale Yellow-Green. 


Aspergillus spp. new to China... 379 


Colonies on CYA at 37 °C after 14 days: growing fast, 48-50 mm in diam., 
plane, centrally umbonate, margins entire, compactly floccose; conidiogenesis 
heavy, Drab Gray; mycelia white, only conspicuous at margins; exudate and 
soluble pigment absent; reverse Pale Yellow-Green to Light Viridine Green. 


ISOLATE EXAMINED: China, Shandong Province, foot of Mount Tai, from soil, 23 July 
2011, Z. Yang (CGMCC AS3.15302). 


PLATE 3. Aspergillus keveii (AS3.15305): A-B. colonies on CYA, CY20S at 25 °C after 14 days; 
C-D. conidiophores; E. conidia. Bar = 10 um 


380 ... Wang 


Aspergillus keveii Varga, Houbraken & Samson PE 3 

Colonies on CA at 25 °C growing moderately, reaching 38-40 mm in diam. 
in 14 days, flat, sparse, floccose, margins fimbriate; conidiogenesis sparse; 
mycelia white; exudate and soluble pigment absent; reverse white. 

Colonies on CYA at 25 °C growing rapidly, reaching 44-47 mm in diam. in 
14 days, deep, margins fimbriate, floccose; conidiogenesis sparse, mostly in the 
central areas, Light Drab; mycelia white with Pale Brownish Vinaceous tinge; 
exudate and soluble pigment absent; reverse Pale Ochraceous-Buff. 

Conidial heads small, radiate; conidiophores borne from aerial hyphae, 
stipes smooth-walled, brown in color, 200-280 x 5-7.5 um; vesicles subglobose, 
brown-colored, 10-18 um in diam., biseriate; metulae short, 3.5 x 2-3 um; 
phialides ampuliform, 7-7.5 x 2-3 um, with short collula; conidia spheroidal, 
conspicuously roughened, 3-3.6(-4) um; Hiille cells not observed. 

Colonies on CY20S at 25 °C growing moderately, reaching 35-38 mm in 
diam. in 14 days, deep, margins fimbriate, floccose; conidiogenesis sparse; 
mycelia white with Pale Grayish Vinaceous tinge; exudate and soluble pigment 
absent; reverse Light Ochraceous-Salmon. 

Colonies on CYA at 37 °C showing no growth after 14 days. 


ISOLATE EXAMINED: China, Shandong Province, foot of Mount Tai, from soil, 23 July 
2011, Z. Yang (CGMCC AS3.15305). 


Aspergillus pseudodeflectus Samson & Mouch. PL. 4 

Colonies on CA at 25 °C growing moderately, attaining 28-30 mm in diam. 
in 14 days, deep, flat, floccose; conidiogenesis absent; mycelia white; exudate 
and soluble pigment absent; reverse white. 

Colonies on CYA at 25 °C fast-growing, reaching 43-45 mm in diam. in 
14 days, deep, convex, floccose; conidiogenesis moderate, Light Drab; mycelia 
white; exudate and soluble pigment absent; reverse Marguerite Yellow to Reed 
Yellow. 

Conidial heads small, radiate; conidiophores borne on aerial hyphae, stipes 
curved and slender, smooth-walled but with occasional concretions, light 
brown in color, (50-)100-180 x 2-3.5 um; vesicles hemispherical, brown- 
colored, 4-7.5 um in diam., biseriate; metulae very short, 3.5 x 2-3 um; 
phialides ampuliform, 7-7.5 x 2-3 um, with long collula up to 3.5 um; conidia 
spheroidal, warty, 2.5-3.6(—4) um; Hille cells not observed. 

Colonies on CY20S at 25 °C growing rapidly, reaching 42-43 mm in diam. 
in 14 days, deep, convex with depression at centers, floccose; conidiogenesis 
moderate, Light Drab to Drab Gray; mycelia white; exudate and soluble pigment 
absent; reverse Reed Yellow. 

Colonies on CYA at 37 °C growing rapidly, reaching 48-50 mm in diam. in 14 
days, flat, annularly and radiately plicate, umbonate lightly in centers, floccose; 


Aspergillus spp. new to China... 381 


% 


ae! 


PiaTE 4. Aspergillus pseudodeflectus (AS3.15306): A-B. colonies on CYA, CY20S at 25 °C after 
14 days; C-E. conidiophores; F. conidia. Bar = 10 um 


382 ... Wang 


conidiogenesis heavy, Light Cinnamon-Drab to Light Drab; mycelia white; 
exudate and soluble pigment absent; reverse Dark Olive to Buffy Brown. 


ISOLATE EXAMINED: China, Shandong Province, foot of Mount Tai, from soil, 23 July 
2011, Z. Yang (CGMCC AS3.1506-3.15310). 


Discussion 
Emericella heterothallica was the first reported heterothallic teleomorph 


associated with the aspergilli. Raper & Fennell (1965) reported the production 
of cleistothecia following the cross of yellow series and reddish orange series. 
Houbraken et al. (2007), however, were unable to reproduce this result. 


A. pseudodeflectus AS 3.15310 
A. pseudodeflectus AS 3.15309 
A. pseudodeflectus AS 3.15307 
0.02 A. pseudodeflectus AS 3.15308 
ie 4 A. pseudodeflectus AS 3.15306 
844. pseudodeflectus NRRL 278 
100}"4. pseudodeflectus NRRL 6135 T 
A. calidoustus CBS 113228 


7) A. calidoustus AS 3.15302 
A. calidoustus CBS 114380 


A. insuetus CBS 107.25 T 
100} 4. minutus NRRL 4876 T 
8884. minutus NRRL 279 


100 A. insuetus CBS 119.27 


99f A. keveii CBS 209.92 T 
99 A. keveii CBS 561.65 
94 A. keveii NRRL 1974 


A. keveii AS 3.15305 


100] A. ustus AS 3.15312 
A. ustus AS 3.15311 

100 A. ustus NRRL 257 T 
A. ustus NRRL 4991 


99 89 A, puniceus NRRL 5077 T 
89 E. heterothallica NRRL 5096 T 
100}"F. heterothallica NRRL 5097 
E. heterothallica AS 3.15313 

‘A. granulosus NRRL 1932 T 

100 ‘A. lucknowensis NRRL 3491 T 

fe A. deflectus NRRL 2206 T 
‘A. elongatus NRRL 5176 T 
‘A. versicolor NRRL 238 T 

‘A. egyptiacus NRRL 5920 T 

A. amylovorus NRRL 5813 T 


100 


PLaTE 5. The NJ tree inferred from the partial sequences of calmodulin gene data set. Bootstrap 
percentages > 70% derived from 1000 replicates are indicated at the nodes. Bar = 0.02 substitutions 


per nucleotide position. 


Aspergillus spp. new to China ... 383 


According to the characters presented, isolate AS3.15313 is a member of the 
reddish orange series. Although E. heterothallica has only been reported rarely, 
our observation of this species in China suggests that, despite its relative rarity, 
it is geographically widely distributed. 

Raper & Fennell (1965) cited six isolates —WB275 (type culture), WB278, 
WB280, WB281, WB1974, WB4876— as typical of the six morphotypes they 
recognized from among the hundreds of A. ustus isolates they had examined. It 
is interesting how predictive of distinct species their observations were. 

Based on partial 6-tubulin, calmodulin, actin genes, and ITS2-5.8S-ITS2 
rDNA sequences, Houbraken et al. (2007) restricted A. ustus to isolates WB275 
and WB280 and designated WB1974 as a new species, A. keveii. Varga et 
al. (2008) included WB281 in their new taxon, A. calidoustus. We observed 
much variation among our isolates under the name of A. ustus sensu Raper 
& Fennell (1965), with prominent differences in the stipe and vesicle shapes 
and dimensions. The growth rate at 37 °C varied greatly, some strains rapid, 
others moderate, still others with no growth at all. Conidiation was also highly 
variable, ranging from abundant to none depending on the isolates and culture 
conditions, and conidial color ranged from grey-green to drab and brown. 
These morphological differences suggest that multiple cryptic taxa may remain 
embedded in A. ustus sensu Raper & Fennell (1965). Phylogenetic analyses of 
three genetic markers (the Nuc ITS1-5.8S-ITS2 rRNA region and the partial 
6-tubulin and calmodulin gene sequences) affiliate our isolates with the four taxa 
included in sect. Usti by Houbraken et al. (2007) and Samson et al. (2011). 


Acknowledgments 

The author would like to thank Dr. J.A. Scott and S.W. Peterson for pre-submission 
review and valuable suggestions. This work was supported by the National Natural 
Foundation of China (no. 31070015). 


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Glass NL, Donaldson GC. 1995. Development of primer sets designed for use with the PCR to 
amplify conserved genes from filamentous Ascomycetes. Appl. Environ. Microbiol. 61: 
1323-1330. 

Hall TA. 1999. Bioedit: a user-friendly biological sequence alignment editor and analysis program 
for Windows 95/98/NTT. Nucl. Acids Symp. Ser. 41: 95-98. 

Houbraken J, Due M, Warga J, Meijer M, Frisvad JC, Samson RA. 2007. Polyphasic taxonomy of 
Aspergillus section Usti. Stud. Mycol. 59: 107-128. http://dx.doi.org/10.3114/sim.2007.59.12 
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of molds from foods. J. Appl. Environ. Microbiol. 37: 959-964. 

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Mycologia 85: 100-107 


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Klich MA. 2002. Identification of common Aspergillus species. CBS Press, Utrecht. 
http://dx.doi.org/10.2307/3760484 

Kozakiewicz Z. 1989. Aspergillus species in stored products. Mycol. Pap., 161: 1-188 

Malloch D. 1981. Moulds their isolation, cultivation and identification. University of Toronto Press, 
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Peterson SW. 2000. Phylogenetic relationships in Aspergillus based on rDNA sequence analysis. 
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and Aspergillus classification. Harwood Academic Publishers, Amsterdam. 

Peterson SW. 2008. Phylogenetic analysis of Aspergillus species using DNA sequences from four 
loci. Mycologia 100: 205-226. http://dx.doi.org/10.3852/mycologia.100.2.205 

Qi Z-T, Kong H-Z, Sun Z-M. 1997. Flora fungorum sinicorum vol. 5: Aspergillus et teleomorphi 
cognati. Science Press, Beijing. (in Chinese) 

Raper KB, Fennell DI. 1965. The genus Aspergillus. Williams & Wilkins, Baltimore. 

Ridgway R. 1912. Color standards and color nomenclature. Publ. by author, Washington D C. 

Samson RA, Varga J, Meijer M, Frisvad JC. 2011. New taxa in Aspergillus section Usti. Stud Mycol., 
69: 81-97. http://dx.doi.org/10.3114/sim.2011.69.06 

Scott J, Malloch D, Wong B, Sawa T, Straus N. 2000. DNA heteroduplex fingerprinting in Penicillium. 
225-236, in RA Samson, JI Pitt (eds). Integration of modern taxonomic methods for Penicillium 
and Aspergillus classification. Harwood Academic Publishers, Amsterdam. 

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Varga J, Houbraken J, Van Der Lee HAL, Verweij PE, Samson RA. 2008. Aspergillus calidoustus sp. 
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White TJ, Bruns T, Lee S, Taylor J. 1990. Amplification and direct sequencing of fungal ribosomal 
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ISSN (print) 0093-4666 © 2012. Mycotaxon, Ltd. ISSN (online) 2154-8889 


MY COTAXON 


http://dx.doi.org/10.5248/120.385 
Volume 120, pp. 385-400 April-June 2012 


Systematics of the Gomphales: 
the genus Gomphus sensu stricto 


ADMIR J. GIACHINI’*, CARLA M. CAMELINI’, MArcrio J. Rossr’, 
CLAUDIO R. F. S. SOARES’ & JAMES M. TRAPPE? 


Universidade Federal de Santa Catarina, Departamento de Microbiologia, 
Imunologia e Parasitologia, Florianopolis, Santa Catarina 88040-970, Brazil 

Oregon State University, Department of Forest Ecosystems and Society, 
Corvallis, Oregon 97331-5752, USA 


*CORRESPONDENCE TO: admir.giachini@ufsc. br 


ABSTRACT — Gomphus sensu lato (Gomphales) was described to include species of 
cantharelloid-gomphoid fungi that had ‘merulioid’ (wrinkled) hymenia and verrucose spores. 
Gomphus sensu stricto is currently characterized by unipileate to merismatoid (composed 
of several pilei) basidiomata, depressed funnel- to fan-shaped pilei, presence of clamp 
connections, and verrucose spores; it has three described species: G. brunneus, G. clavatus, 
and G. crassipes. Basidiomata and spore features are reliable identifiers of Gomphus sensu 
stricto that distinguishes the species from other genera in the Gomphales. 


Key worps — Gloeocantharellus, Phaeoclavulina, Turbinellus 


Introduction 

The genus Gomphus was originally proposed as a segregate from the genera 
Clavaria, Geoglossum, Mitrula, and Spathularia and described as “thickened, 
truncate, smooth, laterally plicate-venose, the pileus weakly developed” 
(Persoon 1797). No species were assigned to the genus when described. The 
first citation of a species for Gomphus came only when Gray (1821) described 
G. clavatus based on Merulius clavatus Pers., a species known to have 
merismatoid (composed of several pilei) basidiomata, orangish brown to violet 
pilei, violet hymenia, and verrucose non-anastomosed ornamented spores. 

From the earliest citations of Persoon (1797) to the latest descriptions of 
Petersen (1971), the systematics and nomenclature of Gomphus have had a 
checkered history. Before Giachini (2004) analyzed the molecular phylogeny 
of 320+ collections of Gomphus sensu lato and related genera in the Gomphales 
and reviewed the systematics and nomenclatural history for this group, species 
of Gomphus sensu stricto were assigned to three different genera: Cantharellus 


386 ... Giachini & al. 


(Fries 1821), Craterellus (Fries 1838), and Neurophyllum (Doassans & Patouillard 
1886). 

The reduced number of distinctive morphological features and (more 
importantly) the lack of molecular data to clarify the systematics of this group 
have contributed to its confused classification. Nevertheless, the significant 
contributions by many taxonomists have led to a better understanding of the 
overall placement of gomphoid fungi. 

Recently Giachini & Castellano (2011) presented a new classification 
for Gomphus sensu lato. Giachini (2004) and Giachini & Castellano 
(2011) emphasized that Gomphus sensu stricto is the only genus in the 
Gomphaceae with strictly violet, lavender-brown, or milky-coffee colored 
hymenia, distinguishing it from other representatives of Gomphus sensu lato 
(Gloeocantharellus, Phaeoclavulina, Turbinellus) characterized by orange, 
brown or greenish olive hymenia. Furthermore, all Gomphus species produce 
clamp connections and verrucose spores. The unique combination of these 
morphological characteristics separates Gomphus from other genera within the 
Gomphales. 

Below we review the important diagnostic characters of Gomphus sensu 
stricto and provide a dichotomous key and complete descriptions of the three 
currently accepted species: G. brunneus, G. clavatus, and G. crassipes. For 
discussion on the nomenclatural history of Gomphus sensu lato see Giachini 
(2004), Giachini et al. (2010), and Giachini & Castellano (2011). 


Materials & methods 


Collections examined 

Collections of Gomphus were obtained from BR, DAOM, FH, K, MICH, NYS, OSC, 
S, SFSU, TENN, and WTU (http://www.nybg.org/bsci/ih/ih.html). Since Dufour (1889) 
did not assign a type for G. crassipes and the only known collections (two) deposited at 
the herbarium RAB were not granted for loan, its description and further notes were 
taken from the relevant literature. 

In addition to dried herbarium material, we examined fresh specimens of G. clavatus 
from northern California, Oregon, and Washington. These were collected mostly in late 
September to mid-December, the usual fruiting season. Therefore, colors for G. clavatus 
are based on fresh and dried material while for G. brunneus are based on dried material 
only. 

After selected collections were photographed and macroscopic features recorded, 
they were dried within 24 h of collection with a forced-air food dehydrator set at 
38-40°C, and deposited at OSC. 

Habitat and fruiting patterns are based on information from the original descriptions 
and/or field observations. 

Macroscopic characterization 

Macroscopic features are based on original descriptions and herbarium notes 

supplemented by our own data on fresh or dried specimens. Features of the pileus, 


Gomphus sensu stricto ... 387 


hymenium, stipe, flesh, odor and taste were recorded whenever possible. Size of 
basidiomata may vary depending on conditions of the fruiting season, so the ranges of 
minimum and maximum sizes are presented as the range for 5-10 collections. Several 
specimens were cut longitudinally through the vertical axis to examine the context 
structure of pileus and stipe. Application of chemicals often used and referred to in the 
literature (Corner 1966, Petersen 1971) in describing species of Gomphus sensu lato 
proved of little value and so are not reported here. 

Microscopic characterization 

Features observed with a compound microscope were described from free-hand 
sections mounted in Melzer’s reagent, 5% KOH, cotton blue, or H,O. Structures were 
measured with an optical micrometer at x1000 magnification, mostly in mountants. 
Hyphal wall thicknesses are recorded as ‘thin’ <0.5 um and ‘thick’ >0.5 um. The pileipellis 
and stipitipellis were examined in surface view. When material was available, dimensions 
were recorded from at least 3 specimens from each of at least 5 collections. Both mature 
and immature specimens were examined for developmental changes. Maturity was 
judged by the relative abundance of fully ornamented spores. 

Spore shape was determined by the length-width ratio of 20-30 randomly selected 
spores (Kirk et al. 2008). Dimensions are given as: minlength-maxlength x minwidth- 
maxwidth, excluding ornamentation and apiculus. Immature or oversized spores were 
not included in the measurements, though variation was noted. 


Taxonomy 

Species are presented in alphabetical order. Descriptions include citation of 
all examined material and the herbarium where the type and other collections 
are deposited. Descriptions are based on notes available with collections, the 
observations made in this study, and literature reports. 


Gomphus Pers., Tent. Disp. Meth. Fung.: 74 (1797) [non Gomphus (Fr.) Weinm.1826]. 
(For synonymy, see Giachini & Castellano 2011:186.) 
TYPE SPECIES: Gomphus clavatus (Pers.) Gray 1821. 


BASIDIOMATA <18 cm tall, erect, unipileate to merismatoid, when merismatoid 
with two to several pilei arising from a single stipe. PILEUs 4-18 cm wide, 
plane to depressed, fan- to funnel-shaped, flexuous, fleshy, dry, glabrous 
(G. brunneus) to subpruinose, covered with brown hyphae that form minute 
separate and distinct patches toward the margin but merge into a continuous 
felty tomentum over the disc (G. clavatus), rosaceous, sordid yellow to orange- 
brown, creamy violet, violet, brown; margin lacerate to crenate. HYMENIUM 
decurrent, wrinkled, generally longitudinally oriented, dichotomous, often 
to rarely anastomosing, reticulate to almost poroid (G. brunneus), especially 
near the stipe apex, more lamellate toward the margin of the pileus, violet, 
vinaceous brown to milky-coffee colored. Stipe <80 mm long, solid, cylindrical 
to tapering downward, generally dilating into the pileus, pale violet to brown, 
nearly black in G. crassipes, often pale red-brown where handled. CONTEXT 


388 ... Giachini & al. 


firm, pliable, often with anastomosing cavities, especially in G. clavatus, white, 
off-white, violet, pale rose, brownish pink on exposure. OpDorR faint to sweet. 
TasTE mild to bitter. SPORE PRINT brown for G. brunneus and G. clavatus, not 
recorded for G. crassipes. 

PILEIPELLIS of scattered to fasciculate, simple or branched, rarely slightly 
inflated hyphae, in G. clavatus with pileocystidia; clamp connections present. 
STIPITIPELLIS of parallel hyphae at surface, interwoven beneath, hyaline, <2 um 
wide; clamp connections present. PILEUS and STIPE CONTEXT of interwoven, 
hyaline hyphae 2.5-6 um wide, generally uninflated except adjacent to the 
clamp connections. HYMENIAL TRAMA of thin- to thick-walled hyphae; clamp 
connections present. SUBHYMENIAL TRAMA of interwoven, hyaline hyphae 
<8 um wide; clamp connections present. Bastp1A <100 um long, 12 um wide, 
clavate, in general with (2-)4 slightly divergent, slightly incurved sterigmata; 
clamp connections present. HYMENIAL CYSTIDIA absent. BASIDIOSPORES 7.5-17 
x 3.5-7.5 um, orange, yellow-brown to dark olive in mass, ellipsoid to obovoid; 
ornamentation verrucose, cyanophilic; apiculus eccentric (not recorded for 
G. crassipes). 

ECOLOGY & DISTRIBUTION: epigeous, solitary to gregarious or caespitose, in 
leaf litter or terrestrial. When gregarious, basidiomata are often separated by a 
few centimeters. Encountered in Africa, Asia, Europe, and North America. 


REMARKS: Defining boundaries for species within Gomphus sensu lato has been 
a constant challenge because of the lack of consistent morphological features. 
Only recently have morphology and molecular techniques combined to provide 
a natural classification for the Gomphaceae. Giachini et al. (2010) and Giachini 
& Castellano (2011) verified by molecular and morphological data that species 
of Gomphus sensu stricto (G. brunneus, G. clavatus, and G. crassipes) are in 
a lineage separate from all other species of Gomphus sensu lato. Accordingly, 
Gomphus sensu stricto is reduced to three species vs. the 35 formerly assigned 
to Gomphus sensu lato. Main features diagnosing Gomphus are the smooth to 
subpruinose pileus, wrinkled or poroid-like hymenium, and clamp connections. 
Species of Gomphus sensu stricto occur across the northern hemisphere and as 
far south in Africa as the Democratic Republic of the Congo. 


Gomphus brunneus (Heinem.) Corner, Ann. Bot. Mem. 2: 116. 1966. Fic. 1 
(For synonymy, see Giachini & Castellano 2011:186.) 
Type: DEMOCRATIC REPUBLIC OF CONGO. Equateur, Binga, April 1928, M. 
Goosens-Fontana 683 (BR A253). 
BASIDIOMATA <9 x 4 cm, unipileate, erect, clavate, truncate at apex. PILEUS 
<4 cm wide, depressed to funnel-shaped, thick in the center, thin toward the 
ascending and somewhat lacerate margin, brown or occasionally more or 
less rosaceous, with paler tones in the center. HYMENIUM wrinkled, reticulate 


Gomphus sensu stricto ... 389 


Fic. 1. Gomphus brunneus. A. Basidioma (bar = 2 cm). B. Basidiospores (bar = 5 um). 


to almost poroid, pale violet to milky-coffee colored. StrPE <80 mm long, 
tapered downward, dilating obconically into the pileus, brown, the base white 
tomentose. CONTEXT pliable, white, then brownish pink on exposure. ODOR 
not recorded. TasTE bitter. SPORE PRINT brown. 

PILEIPELLIS of scattered to fasciculate, simple hyphae; clamp connections 
present. STIPITIPELLIS of hyaline hyphae <2 um wide; clamp connections 
present. PILEUs and STIPE CONTEXT of interwoven, hyaline hyphae 2.5-6 um 
wide; clamp connections present. HYMENIAL TRAMA of thin-walled hyphae; 
clamp connections present. SUBHYMENIAL TRAMA Of slightly thickened, hyaline 
hyphae 3-7 um wide, with refractive walls; clamp connections present. BASIDIA 
50 x 8 um, with 2-4 sterigmata. HYMENIAL CyYSTIDIA absent. BASIDIOSPORES 
7.5-10 x 3.5-5 um, yellow-brown in mass, ellipsoid to obovoid; ornamentation 
of cyanophilic warts arranged more or less in longitudinal rows; apiculus 
rounded, eccentric. 

ECOLOGY & DISTRIBUTION terrestrial, in leaf litter, on floor of dry forests of 
Cameroon (Roberts 1999), the Democratic Republic of Congo, and Uganda 
(Corner 1966, Roberts 1999). 


ADDITIONAL SPECIMENS EXAMINED: UGANDA: Gunda Forest, May 1918, T:D. Maitland 
275 (as Gomphus clavatus var. parvisporus; K 57307). 


390 ... Giachini & al. 


REMARKS: Gomphus brunneus is distinguished by its simple basidiomata with 
smooth pilei, weakly wrinkled hymenia, clamped hyphae, and comparatively 
small basidiospores. Its morphology, distribution, and molecular profile make 
it unique (Giachini 2004, Giachini et al. 2010, Giachini & Castellano 2011). 

Only two G. brunneus herbarium collections were available for examination. 
After studying the type, Roberts (1999) considered G. clavatus var. parvisporus 
from Uganda (described by Corner 1966 and said to occur in Europe and the 
western USA) a synonym of G. brunneus. Both species are macroscopically 
similar with unusually small verrucose spores and are known only from 
equatorial Africa. Heinemann (1958, 1959) reported two collections from 
forests of the Democratic Republic of Congo, Africa. Corner (1966) reported 
one collection (as Gomphus clavatus var. parvisporus) from Cameroon, while 
Roberts (1999) cited two collections from Cameroon obtained in 1996 and 
reported abundant basidiomata from the same location on the following year. 
Otherwise, the species appears to be restricted to the Democratic Republic of 
Congo and Uganda. 

For several decades Cantharellus pseudoclavatus A.H. Sm. (Smith & Morse 
1947) was classified under Gomphus sensu lato (Corner 1966). After analyzing 
the type collection (MICH 6916), however, Giachini (2004) concluded that it 
better fit Cantharellus. The main morphological character justifying its removal 
from Gomphus sensu lato was the presence of 8-spored basidia, a feature not 
found elsewhere in Gomphus but present in Cantharellus. Petersen (1971) had 
previously transferred the species to Pseudocraterellus, a genus which also lacks 
8-spored sterigmata. Although Corner (1966) suggested Gomphus clavatus var. 
parvisporus occurs in Europe and is common in western USA, he mentioned 
only specimens obtained in Africa in his original description of that variety. 
Cantharellus pseudoclavatus is rare under conifers of the western USA and is 
definitely distinct from G. brunneus. 


Gomphus clavatus (Pers.) Gray, Nat. Arr. Brit. Pl. 1: 638. 1821. Fig, 2 
(For synonymy, see Giachini & Castellano 2011:187.) 

BASIDIOMATA S17 cm tall, unipileate at first and then merismatoid with 
<15 subpilei. Prteus <15 cm wide, the surface flat, subundulate, fan-shaped, 
glabrous to covered with brown hyphae that form minute separate and distinct 
patches toward the margin but merge into a continuous felty tomentum 
over the disc, orangish brown to overall creamy violet to dark violet; margin 
crenate. HyMENIUM surface wrinkled, generally longitudinally oriented, with 
or without discrete folds or pits, bright violet at the margin and junction with 
stipe and overall when immature, at maturity covered with spores and then 
paler (vinaceous brown). STIPE tomentose to hispid toward the base, glabrous 
above and there blending to pale violet, white at the base and where covered 


Gomphus sensu stricto ... 391 


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Dr. Robert Thoma$ anMargarett 


Fic. 2. Gomphus clavatus. A-B. Basidiomata. C. Spores (bar = 10 um). 


392 ... Giachini & al. 


(soil, debris), often pale red-brown where handled. CoNnTEXT often with 
anastomosing cavities, violet around those, off-white to pale rose elsewhere. 
Opor faint or none. TasTE mild. SPORE PRINT brown. 

PILEIPELLIS often crowded, thin-walled, scattered to fasciculate, with simple 
or branched hyphae, usually incrusted apically, with pileocystidia 3-4.5 um 
wide, rarely slightly inflated, <5.5 um, protruding 50-120 um from the surface; 
clamp connections present. Hyphae and bases of pileocystidia often with 
brown amorphous deposits. STIPITIPELLIS of periclinal and parallel, hyaline 
hyphae 1.5-2 um wide at the surface, becoming interwoven beneath, with few 
projecting hyphae where stipe is glabrous, or fascicles of crowded hyphae where 
stipe is hispid; clamp connections present. PILEUS and STIPE CONTEXT of thin- 
to slightly thick-walled, generally oriented on the long axis of the basidioma 
but profusely interwoven, hyaline hyphae 2.5-6 um wide, generally uninflated 
except adjacent to the clamp connections, where it is slightly thick-walled 
(<1 um thick) and <12 um wide. HyMENIAL TRAMA hyphae more loosely 
interwoven than the rest of the context, undifferentiated from the sUBHYMENIAL 
TRAMA. BASIDIA 60-95 x 8.5-12 um, clavate, collapsing after spore discharge, 
hyaline, with (2-)4 slightly divergent, slightly incurved sterigmata <10 um 
long; clamp connections present at base. HYMENIAL CYSTIDIA not observed. 
BASIDIOSPORES (9-)10-15(-17) x 4-7.5 um, orange in Melzer’s reagent, 
dark olive in mass (KOH), ellipsoid to obovoid, contents weakly cyanophilic; 
ornamentation of cyanophilic warts, usually in discrete, raised patches that give 
an undulate appearance to the spore surface; apiculus prominent, eccentric. 

ECOLOGY & DISTRIBUTION: solitary, gregarious to caespitose, when gregarious 
often separated by a few centimeters, terrestrial. Generally in conifer forests and 
suggested to form mycorrhizae with species of Abies (Pantidou 1980) and Picea 
(Agerer et al. 1998). Known from Austria (Petersen 1971), Canada (East and 
West, Petersen 1971), China (Corner 1966), Czech Republic (Kluzak 1994), 
France (Doassans & Patouillard 1886), Greece (Petersen 1971), Italy (Petersen 
1971), Japan (Corner 1966), Lithuania (Urbonas et al. 1990), Mexico (Petersen 
1971), Pakistan (Corner 1966), Poland (Adamczyk 1996), Russia (Bulakh 1978, 
Bulakh & Govorova 2000), Spain (Fernando Sanchez, pers. comm., 10 Oct. 
1999), Sweden, Switzerland (Petersen 1971), Turkey (Sesli 1997), and the USA 
(especially northwestern/northeastern and into the Appalachian Mountains). 
Loans were requested from herbaria located in most of these countries but 
several did not respond, so our examination of specimens is restricted to those 
listed under collections examined. 

ADDITIONAL SPECIMENS EXAMINED: CANADA. BRITISH COLUMBIA, Vancouver 
Island, 15 October 1962, M. Pantidou (DAOM 91369). Quebec, Duchesnay, 26 Aug. 
1938, H.S. Jackson (DAOM 8624). SWEDEN: Uppsala, Sédermanland, Handen, 4 


Sep. 1998, L. Kerwién (S); Fiby Urskog, 20 Sep. 1998, A. Taylor (S). UNITED STATES. 
CALIFORNIA, DEL NorTE Co., Del Norte Coast Redwoods State Park, 9 Dec. 1999, M.A. 


Gomphus sensu stricto ... 393 


Castellano (OSC 97685); Jedediah Smith Redwoods State Park, 17 Nov. 1961, D. Isely 
1479 (WTU), and 12 Nov. 1966, H.D. Thiers (SFSU 17740); Siskiyou Nat. Forest, Smith 
River, 30 Nov. 1937, A.H. Smith 9243 (MICH); EL Dorapo Co., Pleasant Valley, 22 
October 1967, D.E. Stuntz 14410 (WTU); MENDocINo Co., 5 Nov. 1961, D. Largent 
71 (SFSU); Aleuria Glenn, 14 Nov. 1967, R.H. Petersen (TENN 33256); Aleuria Glenn, 
along Rd. 404, 25 October 1986, M. Seidl 2120 (WTU)); Jackson State Forest, 5 Nov. 
1967, R.H. Petersen and H.D. Thiers (TENN 33174), and 8 Dec. 1999, A.J. Giachini, M.A. 
Castellano and E. Nouhra (OSC 97679, 97680, 97681, 97682, 97683, 97684). IDAHO, 
BONNER Co., Priest River, Binarch Creek, 9 Sep. 1966, R.H. Petersen (TENN 32166); 
Priest River Experimental Forest, Canyon Creek Road, 11 Nov. 1939, A.H. Slipp 621 
(MICH). MassAcHusETTS, Essex Co., Gloucester, Aug. 1877, unnamed collector 
(FH). NEw YorK, SARATOGA Co., Ballston Lake, Branch, 1879, C.H. Peck (holotype of 
Cantharellus brevipes; NYS). OREGON, BENTON Co., Siuslaw Nat. Forest, Mary’s River, 
24 October 1999, S. Ashkannejhad (OSC 97622); CLacKAMAS Co., Wemme, 23 Sep. 
1946, A.H. Smith 23644 (MICH); Coos Co., South Slough, 23 Sep. 1985, C. Ardrey 800 
(WTU), and 3 Sep. 1992, C. Ardrey 1806 (WTU); LANE Co., Cougar Reservoir, Road 
19, 27 Nov. 1999, A.J. Giachini (OSC 97671, 97672, 97673, 97674, 97675, 97676, 97677); 
Eugene, 30 October 1999, unnamed collector (OSC 97646); H. J. Andrews Experimental 
Forest, Lookout Mt., Rd. 1506, 23 October 1999, A.J. Giachini (OSC 97616, 97620, 97669, 
97670, 97678); H. J. Andrews Experimental Forest, 23 October 1999, A.J. Giachini (OSC 
97617), 5 Nov. 1999, A.J. Giachini (OSC 97656), and 7 Nov. 2000, Y. Yano (OSC 97694); 
Oakridge, Road 1934, 15 Nov. 1999, A.J. Giachini (OSC 97668); Willamette Nat. Forest, 
25 October 1999, C. Lefevre (OSC 97623); LINN Co., Sweet Home, 4 Nov. 2000, A.J. 
Giachini (OSC 97696, 97698); Willamette Nat. Forest, Pamela Lake Trail head, 2 Nov. 
1999, A.J. Giachini (OSC 97624); MARION Co., Detroit Lake, Road 46, 15 Nov. 1999, A.J. 
Giachini (OSC 97667). WASHINGTON, CHELAN Co., Lake Wenatchee, 7 Nov. 1980, D.E. 
Stuntz 21131 (WTU); CLALLAM Co., Morse Creek camp, 17 October 1942, D.E. Stuntz 
1245 (WTU); Olympic Mountains, 30 October 1931, H.S. Hotson (WTU); KinGs Co., 
Snoqualmie Nat. Forest, Asahel Curtis Nature Trail, 4 Aug. 1992, G.R. Walker 10600 
(WTU); Lewis Co., Mt. Rainier Nat. Park, Bumping Lake, October 1937, D.E. Stuntz 
759 (WTU); PreRcE Co., Mt. Rainier Nat. Park, Lower Tahoma Lake, 23 Sep. 1948, 
D.E. Stuntz 4732 (WTU); Old Tahoma campground, 23 Sep. 1960, D.E. Stuntz 12000 
(WTU); Mt. Rainier National Park, Tahoma Creek, 18 Aug. 1948, D.E. Stuntz and A.H. 
Smith 4003 (WTU); Mt. Rainier Nat. Park, Tahoma Creek, 23 Sep. 1960, D. Isely 1257 
(WTU). 


REMARKS: Gomphus clavatus has traditionally been placed in a monotypic 
subgenus (Gomphus subg. Gomphus) based on morphological features (Corner 
1966). However, molecular studies by Giachini (2004) and Giachini et al. (2010) 
support its placement together with G. brunneus and G. crassipes in Gomphus 
sensu stricto and suggest it to be phylogenetically closely related to species of 
Gloeocantharellus. Petersen (1971) noted that these two genera share several 
morphological similarities, the most striking of which is that both exhibit the 
same reaction to 10% KOH applied to the basidioma pileus. In reference to 
G. clavatus Petersen (1971) stated, “the normal color immediately becomes a 
most unique salmon-yellow, almost precisely the normal color of the pileus 
and stipe of Gloeocantharellus purpurascens (Hesler) Singer.” The pileus surface 
tomentum of G. clavatus is composed of pileocystidia, just as in G. purpurascens, 


394 ... Giachini & al. 


and, even though the pileocystidia of the two species differ in shape and 
orientation, they have a similar brown agglutinating substance. Despite these 
similarities, the molecular evidence shows Gomphus and Gloeocantharellus to 
be in distinctly different, but related lineages (Giachini et al. 2010). 

Other taxa in the Gomphaceae share the merismatoid habit of G. clavatus, 
notably Phaeoclavulina grandis (Corner) Giachini, P. guadelupensis (Pat.) 
Giachini, P. subclaviformis (Berk.) Giachini, and P. viridis (Pat.) Giachini and 
Gloeocantharellus dingleyae (Segedin) Giachini, G. novae-zelandiae (Segedin) 
Giachini, and G. pallidus (Yasuda) Giachini. 


Gomphus crassipes (L.M. Dufour) Maire, in Maire & Werner, Mém. Soc. Sci. Nat. 
Maroc 45: 81 (1937). FIG. 3 
(For synonymy, see Giachini & Castellano 2011:187.) 
Lectotype designated here, Pl. 13, in L.M. Dufour, Rev. Gen. Bot. 1: 357-358. 1889. 


BASIDIOMATA <18 cm tall, unipileate or occasionally merismatoid. PILEUs 4-18 
cm wide, plane to slightly depressed in the center, funnel-shaped to fan-shaped, 
non hygrophanous, sordid yellow to orangish brown; margin subundulate. 
HyMENIumM decurrent, wrinkled, occasionally anastomosing, dichotomous, 
violet. Stipe 40-70 x 25-45 mm, cylindrical to slightly tapering downward, 
almost entirely underground, central or lateral, nearly black. CONTEXT firm, 
pale violet, marbled in the stipe with violet tones. ODOR sweet. TASTE not 
recorded. SPORE PRINT color not recorded. 

All HyPHAE 5-8 um wide; clamp connections present. Basrp1a 70-100 x 
8-10(-12) um, clavate, with (2-)4 straight sterigmata. HYMENIAL CYSTIDIA 
absent. Bastip1osporEs (11-)13-15(-17) x 5.5-6(—7) um, pale orange in mass, 
ellipsoid to obovoid; ornamentation of cyanophilic, fine warts; data on apiculus 
not recorded (Dufour 1889, Maire 1914). 

ECOLOGY & DISTRIBUTION: on the ground, generally grouped, in conifer 
forests in Algeria (Dufour 1889, Maire 1914, Maire & Werner 1937), Morocco 
(Malencon 1958), and Spain (Miguel A. Pérez-De-Gregorio i Capella, pers. 
comm., 5 Jan 2004). 


REMARKS: As the only known collections are at herbarium RAB in Morocco, 
and our loan request was denied due to the material’s fragile condition, our 
description is based on Dufour (1889), Maire (1914), and Maire & Werner 
(1937). Dufour (1889), who did not cite any collection or herbarium for the 
type species, stated that “cette espéce est assez voisine du Cantharellus brevipes 
Peck” As C. brevipes is a synonym of G. clavatus, in effect Dufour related 
G. crassipes to G. clavatus, with similar macro- and microscopic characteristics, 
differing only by the violet to marbled violet color of the context, which is 
violet, off-white to pale rose in G. clavatus. However, its overall spore size and 


Gomphus sensu stricto ... 395 


Fic. 3. Gomphus crassipes. 
A. Basidiomata [from Maire (1914: Pl. VII.2)]. 
B. Dried basidiomata [modified from Dufour (1889; Pl. 13), with permission]. 


396 ... Giachini & al. 


geographic distribution suggest that G. crassipes is distinct from G. clavatus 
(Giachini et al. 2010). 

Only a few other references to G. crassipes are observed in the literature. 
Maire (1914), who transferred Dufour’s C. crassipes to Neurophyllum, provided 
a detailed description and excellent drawing of its basidiomata and spores 
(Maire 1914) and subsequently (Maire & Werner 1937) transferred the species 
to Gomphus. Later Malencon (1958) called attention to the putative relationship 
of G. crassipes to the coral fungi, especially to Ramaria, but observed that Maire’s 
drawings were not good representations of what Dufour (1889) had described 
as C. crassipes and that in particular the were not those suggested by Dufour. 
This species “pulls too much into the yellows and reds” according to Malencon 
(1958), who described G. crassipes as having a pale ocher to rose pileus surface. 
However, as seen for other species of Gomphus sensu lato, (e.g., G. clavatus) 
the color of both pileus and hymenium surface can vary considerably. For 
G. clavatus, for example, the pileus surface can be orangish brown to overall 
creamy violet to dark violet. It is possible that Maire (Maire & Werner 1937) 
observed a color variant of G. crassipes. 

Because the designated lectotype does not adequately characterize the 
species, an epitype is desirable. However, we refrain from designating an epitype 
since no herbarium specimen is available and Malencon (1937) challenged 
the Maire’s (1914) excellent illustration. A fresh collection (preferably from 
Morocco) that can provide DNA sequences would be an ideal candidate for 
epitypification. 


Key to species 


la. Basidiomata initially unipileate, then becoming merismatoid with <15 subpilei; 
pileus fan-shaped, orangish brown, creamy violet to dark violet; hymenium 
wrinkled, violet, vinaceous brown, lavender-brown; 
ASTAENITO PSs aN OFT AMTICRICAS. 5 jc a-seslin bee hart wast ecare wat siaet ea otsee eee one G. clavatus 


1b. Basidiomata unipileate to merismatoid; pileus depressed, funnel-shaped, 
occasionally fan-shaped, brown to rosaceous, sordid yellow to orangish brown; 
hymenium wrinkled, reticulate to almost poroid, violet to milky-coffee colored; 
PNT CAs DAIS Jap ctrae tons ee oa escalate ted et A Ie as mete doses Ape datetents Z 


2a. Spores 7.5-10 x 3.5-5 uum, verrucose; basidiomata unipileate; pileus brown or 
occasionally rosaceous; hymenium slightly wrinkled, reticulate to almost poroid, 
pale violet to milky-coffee colored; 
Cameroon, Democratic Republic of Congo, Uganda .............. G. brunneus 


2b. Spores (11-)13-15(-17) x 5.5-6(-7) um, finely verrucose; basidiomata 
occasionally merismatoid; pileus sordid yellow to orangish brown; hymenium 
wrinkled, violet; 
I SETIa, -WIOKOCES, ANC SPallics J.:.8 ce Tank rath a tise asrablatstoreaaek eh earasen G. crassipes 


Gomphus sensu stricto ... 397 


Discussion 

Gomphus was originally described to include cantharelloid-gomphoid 
species that produced basidiomata resembling those of Cantharellus. The recent 
molecular analyses of Giachini et al. (2010), which sharpened generic and 
species concepts within the Gomphaceae and revealed Gomphus sensu lato as 
non-monophyletic, support redistribution of those into Gomphus sensu stricto, 
Gloeocantharellus, Phaeoclavulina, and Turbinellus. Only three original species 
are retained within Gomphus sensu stricto — G. brunneus characterized by 
somewhat funnel-shaped pilei and G. clavatus and G. crassipes, characterized 
by fan- to funnel-shaped pilei. 

Gomphus clavatus, widely distributed in the northern hemisphere, is 
believed to form ectomycorrhizae with species of Abies (Pantidou 1980) and 
Picea (Agerer et al. 1998). It is the most easily identified of the three species, 
due primarily to the size and color of its basidiomata. Since the development 
and implementation of the Northwest Forest Plan in the United States (USDA/ 
USDI 1994a,b, 2000, 2001), G. clavatus has been considered rare and potentially 
restricted to old-growth forests of the Pacific Northwest (PNW), requiring all 
federal and state land to be surveyed for the species before any management 
activity could be approved. 

Both G. brunneus and G. crassipes are known only from a few collections 
from Africa and Spain, as noted above. 

Morphologically Gomphus sensu stricto is readily separated from the other 
gomphalean genera based on the violet, violet-brown to orangish brown colors 
and fan- to somewhat funnel-shaped basidiomata pilei. 

Species of Gomphus sensu stricto most resemble those of the resurrected 
genus Phaeoclavulina (Giachini & Castellano 2011), which share fan- to funnel 
shaped pilei and clamp connections. However, they differ based on spore 
ornamentation, with most Phaeoclavulina species diagnosed by echinulate, 
subreticulate, or reticulate spores in contrast to the strictly verrucose spores of 
Gomphus. 

Even though all Turbinellus species were assigned to Gomphus until 
recently, they share only few morphological characters, most notably spore 
ornamentation. They differ in basidiomata shape, color, and presence of clamp 
connections (absent in Turbinellus). 

Gomphus and Gloeocantharellus occupy an important position in the 
evolution of the fungi in the Gomphales. Molecular analyses (Giachini et al. 
2001, Giachini et al. 2010, Humpert et al. 2001), indicate Gloeocantharellus and 
Gomphus as ancestral to all other genera within the Gomphales. The two genera 
share spore shape and ornamentation and (for some Gloeocantharellus species) 
clamp connections. 

Unavailability of samples restricted more in-depth evolutionary inferences 
of Gomphus to other genera in the Gomphales. Additional sampling, especially 


398 ... Giachini & al. 


for G. crassipes, is necessary to determine whether subgeneric arrangements 
exist within Gomphus sensu stricto and to infer higher-level relationships to 
other members of the Gomphales. A complete discussion on the evolutionary 
relationships and implications of this taxonomic classification in relation to 
other members of the Gomphales can be found in Giachini (2004), Hosaka et 
al. (2006), and Giachini et al. (2010). 


Acknowledgments 

This research was partially supported by the Forest Mycology Team (Pacific Northwest 
Research Station, Forest Service, US Department of Agriculture, Corvallis, Oregon). 
AJG thanks Conselho Nacional de Desenvolvimento Cientifico e Tecnolégico (CNPq) 
of the Brazilian Ministry of Education for the doctorate fellowship. Special thanks to 
the following herbaria for providing specimens: Herbarium of the Jardin Botanique 
National de Belgique - Meise (BR), National Mycological Herbarium —- Ottawa (DAOM), 
Farlow Herbarium of Cryptogamic Botany - Cambridge (FH), Royal Botanic Gardens 
Herbarium - Kew (K), University of Michigan Fungus Collection - Ann Arbor (MICH), 
Herbarium of the New York State Museum - Albany (NYS), Oregon State University 
Herbarium - Corvallis (OSC), Herbarium of the Swedish Museum of Natural History 
— Stockholm (S), Harry D. Thiers Herbarium at San Francisco State University - San 
Francisco (SFSU), University of Tennessee Herbarium - Knoxville (TENN), and the 
University of Washington Herbarium - Seattle (WTU). Also thanks to Dr. Andy Taylor 
for specimens of G. clavatus from Sweden. Boleslaw Kuznik, Michael Wood, Robert 
Thomas, and Margaret Orr are acknowledged for permission to reproduce photographs 
and drawings. We are grateful to Drs. Richard E. Baird, Efrén G. Cazares, and Shaun 
Pennycook for critically reading the manuscript. 


Literature cited 

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AOO ... Giachini & al. 


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USDA/USDI [U.S.D.A. Forest Service; U.S.D.I. Bureau of Land Management]. 2001. Record of 
decision and standards and guidelines for amendments to the survey and manage, protection 
buffer, and other mitigation measures standards and guidelines. Portland, OR. 


ISSN (print) 0093-4666 © 2012. Mycotaxon, Ltd. ISSN (online) 2154-8889 


MYCOTAXON 


http://dx.doi.org/10.5248/120.401 
Volume 120, pp. 401-405 April-June 2012 


Myxomycetes from China 15: 
Arcyria galericulata sp. nov. 


Bo ZHANG’, T1AN-Hao LI’, QI WANG’ & Yu LI’* 


‘Engineering Research Center of Chinese Ministry of Education for Edible and Medicinal Fungi & 
?Institute of Agricultural Modernization, 
Jilin Agricultural University, 2888 Xincheng Street, Changchun City, P. R. China 

* CORRESPONDENCE TO: yuli966@126.com 


ABSTRACT — A new species, Arcyria galericulata, is described and illustrated with scanning 
electron micrographs. This species is characterized by a helmet-like (galeiform) structure at 
the top of the sporocarp. Holotype and isotype specimens are deposited in the Herbarium of 
Mycological Institute of Jilin Agricultural University (HMJAU), Changchun, China. 


Key worps — SEM, taxonomy, Trichiales 


Introduction 

Arcyria is a common and important genus of Trichiaceae. Since Wiggers 
established the genus in 1780, more than 55 species have been reported (Kirk 
et al. 2008, Lado 2001, 2005-12), of which 29 species have been reported in 
China (Chen & Li 1999; Chou 1937; Li & Li 1989; Li et al. 1993; Liu et al. 2002; 
Wang & Li 1995, 1996, 2006). A new species found on bark surface of dead 
log in Jingyuetan National Forestry Park, Jilin province, China, in July, 2009 is 
described and illustrated below. 


Materials & methods 

The fruiting bodies and microscopic structures of the new species were examined 
by light and scanning electron microscopes (Martin & Alexopoulos 1969; Li et al. 
1993) and compared with other morphologically similar Arcyria species. Permanent 
slides are mounted in Hoyer’s medium (Martin & Alexopoulos 1969). Coloured slides 
were prepared according to Robbrecht (1974) by spreading capillitium in a drop of 
94% alcohol, determining colour after one minute, and then mounting in Hoyer’s. 
Colour terms are given according to Anonymous (1969). Voucher specimens are 
deposited in the Herbarium of Mycological Institute, Jilin Agricultural University 
(HMJAU). 


402 ... Zhang & al. 


AccV Spot Maqn Det WD Exp /-——-+{ 500m 
150kV30 38x SE 1001 


AccV Spot Maqn Det bs = : 
15.0KY 3.0 308K SE~, 1 OmIaRRIARS 
& J o 


Acc.V Spot Maan." Det pe . AA. AccV Spot Maqn- ‘Det WD Exp 
15.0 kV 3:0 5000x., | =e y § Rats i a 2 1bO kV 3.0 20000x SE 10.1 1, 


eee —_ avis head! ed " i si Pa 


PiaTE 1. Arcyria galericulata (Isotype): A, fruiting bodies. B, a complete, expanded sporocarp. 
C, galeiform structure at the top of the sporocarp and part of expansion of the capillitium. 
D, part of the stalk, showing longitudinal striations, and part of calyculus and sporocarp. E, outer 
surface of a galeiform structure marked with regular circular depressions. F, warted interior of a 
depression. 


Taxonomy 


Arcyria galericulata B. Zhang & Yu Li, sp. nov. PLATE 1-2 
MycoBank MB564122 


Differs from Arcyria papilla by its smaller sporocarp with a larger helmet-like 
protuberance and larger spores. 


TypE—China, Jilin province, Jingyuetan National Forestry Park, on the bark of a dead 
log, 26 July 2009, Zhang Bo 0628 (Holotype HMJAU10244; isotype HMJAU10245). 


EryMoLocy—galericulata (Latin) = with a helmet-like covering, in reference to the 
galeiform protuberance at the top of the sporocarp. 


Arcyria galericulata sp. nov. (China) ... 403 


SPOROCARPS aggregated or united in clusters of 3-8 (with fused stalks), stalked, 
erect, 1-1.5 mm in total height, 2-2.2 mm after expansion of the capillitium. 
Sporocarp obovoid or shortly cylindrical, fawn to snuff brown, fading to brown. 
HyYPOTHALLUS common to a group of sporocarps. STALK 0.8-1.0 mm long, dark 
brown by transmitted light, filled with spore-like cells, cells 18-20 um diam. at 
the base of stalk, subglobose. PERrp1uM single, membranous, persistent, with a 
helmet-like structure at the top of each sporocarp, outer surface of the structure 
marked with regular circular depressions with warted interiors. CALYCULUS 
translucent, saucer-shaped, light, yellow to colourless by transmitted light, 
inner surface with many prominent warts, low irregular ridges forming a net 
with small protuberances. COLUMELLA absent. CAPILLITIUM tubular, elastic, 
branched and anastomosed, pale orange yellow by transmitted light, 3 um diam. 
without ornamentations, 5-6 um diam. with ornamentations, firmly attached 
to the calyculus, decorated with many cogs, half-rings and rings with irregular 
edges, sometimes with a faint reticulation. Sporgs free, yellowish brown in 
mass, yellowish pale to colourless by transmitted light, 6-8 um diam., densely 
warted, with scattered groups of more prominent warts. 


ComMENTSs: Arcyria galericulata has a galeiform structure at the top of the 
sporocarp. Only one other Arcyria, A. papilla Ejale & L.S. Gill (Ejale & Gill 
1992), has a similar papillate structure at the top of the sporocarp but the 


Acc Spot Magn~ Det WD Pe —— ¥ AccV Spot Magn 
15.0kV 3.0 10000x SE 10.3 1 15.0 kV 3.0 @500x 


Acc.V Spot Maqn ID —_— - a , Ace.V Spot M 
15.0kV 3.0 10000x SiERRIGaie of y 1b.0kKV 3.0 10) 80 
4 =. 


PLaTE 2. Arcyria galericulata (Isotype): A, part of calyculus, showing many prominent warts and 
low irregular ridges forming a net with small protuberances. B-C, part of capillitium, showing the 
ornamentation. D, spores. 


404 ... Zhang & al. 


papilla is smaller, the sporocarp is larger (about 4.4 x 1.0 mm after capillitial 
expansion), the capillitium is marked with spines occasionally interrupted 
by rings that form characteristic triangular loops, and the spores are smaller 
(~5 um diam.) and double-walled. 

The capillitial ornamentation of A. galericulata resembles that of A. affinis 
Rostaf., A. helvetica (Meyl.) H. Neubert et al., and A. ferruginea Saut. Arcyria 
affinis is distinguished by its reddish brown sporocarps (about 3-4 mm after 
capillitial expansion), a capillitium broken away leaving only a few ends attached 
to the stalk apex, and larger spores (about 7-9 um in diam.) (Rostafinsky 
1875). Arcyria ferruginea differs in its shorter stalk (about 0.2-0.6 mm long), 
capillitium tube 5-8(-10) um in diam., and larger spores (9-12 um in diam.) 
(Sauter 1841), while A. helvetica is differentiated by a deeply funnel-shaped 
calyculus and capillitium only attached to the calyculus at the center (Neubert 
et al. 1989). 


Acknowledgments 

We express our deep appreciation to Prof. Guozhong Lit (Dalian Nationalities 
University, P.R. China) and Prof. Uno Eliasson (University of Gothenburg, Sweden) for 
their valuable suggestions in peer-reviewing this manuscript. We thank Dr. Pu Liu, Jilin 
Agricultural University for correcting the manuscript. The study was supported by the 
fund from Ministry of Agriculture of China. 


Literature cited 

Anonymous. 1969. Flora of British Fungi: colour identification chart. Royal Botanic Garden 
Edinburgh, H.M. Stationery Off. 6 p 

Chen SL, Li Y. 1999. A preliminary report on the myxomycetes from Zhangjiajie, Hunan Province. 
J. Wuhan. Bot. Res. 17(3): 217-219. 

Chou ZH. 1937. Notes on myxomycetes from China. Bull. Fan. Men. Inst. Biol. 7: 257-278. 

Ejale UA, Gill LS. 1992. Two new species of myxomycetes from southern Nigeria. Acta Mycologica 
27(2): 267-269. 

Kirk PM, Cannon PF, Minter DW, Stalpers JA. 2008 Ainsworth & Bisby’s dictionary of the fungi. 
10th Ed. CAB International, Wallingford 771 p. 

Lado C. 2001. Nomenmyx: a nomenclatural taxabase of myxomycetes. Cuadernos de Trabajo de 
Flora Micoldgica Ibérica 16. 224 p. 

Lado C. 2005-2012. An online nomenclatural information system of Eumycetozoa. 
http://www.nomen.eumycetozoa.com (Consulted April 15, 2012). 

Li Y, Li HZ. 1989. Myxomycetes from China I: A checklist of Myxomycetes from China. Mycotaxon 
35(2): 429-436. 

Li Y, Chen SL, Li HZ. 1993. Myxomycetes from China X: Additions and notes to Trichiaceae from 
China. Mycosystema 6: 107-112. 

Liu CH, Yang FH, Chang JH. 2002. Myxomycetes of Taiwan XIV: Three new records of Trichiales. 
Tainwainia 47(2): 97-105. 

Martin GM, Alexopoulos CJ. 1969. The myxomycetes. University of Iowa Press. Iowa. 561 p. 
http://dx.doi.org/10.2307/1218569 


Arcyria galericulata sp. nov. (China) ... 405 


Neubert H, Nowotny W, Baumann K. 1989. Myxomyceten aus der Bundesrepublik Deutschland 
V. Carolinea 47: 25-46. 

Robbrecht E. 1974. The genus Arcyria Wiggers in Belgium. Bull. Jard. Bot. Nat. Belg. 44: 303-353 
http://dx.doi.org/10.2307/3667676 

Rostafinsky JT. 1875. Sluzowce monografia. Pamietn. Towarz.Nauk. Sci. Paryzu. 6(1): 216-432. 

Sauter AE. 1841. Beitrage zur Kenntnis der Pilz-Vegetation des Ober-Pinzgaues in Herzogthume 
Salzburg. Flora 24: 305-320. 

Wang Q, Li Y. 1995. Two new varieties of Arcyria. J. Jilin Agri. Univ. 17(4): 83-85. 

Wang Q, Li Y. 1996. A new species of Arcyria myxomycetes. Bull. Bot. Res. 16(2): 179-181. 

Wang Q, Li Y. 2006. Trichiales in China. Institute of Science Publish. 134p. 


ISSN (print) 0093-4666 © 2012. Mycotaxon, Ltd. ISSN (online) 2154-8889 


MY COTAXON 


http://dx.doi.org/10.5248/120.407 
Volume 120, pp. 407-413 April-June 2012 


New records of Puccinia species on Poaceae 
from Fairy Meadows, Pakistan 


N.S. AFSHAN”™, A.N. KHALID? & A.R. NIAZI’ 


™Centre for Undergraduate Studies & *Department of Botany, 
University of the Punjab, Quaid-e-Azam Campus, Lahore, 54590, Pakistan 
*CORRESPONDENCE TO: pakrust@gmail.com 


AsBsTRACT — During a survey of rust fungi of Fairy Meadows, Puccinia brachypodii var. 
major on Poa attenuata and P. substriata var. indica on Pennisetum orientale were reported 
for the first time. These new rust fungi records bring to 70 the number of Puccinia species 
reported on Poaceae from Pakistan. Puccinia brachypodii var. poae-nemoralis and P. poarum 
are also reported for the first time from Fairy Meadows. 


Key worps — Anthoxanthum odoratum, graminicolous rust, Hunza 


Introduction 

This paper continues our study of graminicolous rust fungi from Pakistan. 
Previously, about 100 species of graminicolous rust fungi including 68 taxa of 
Puccinia have been reported from Pakistan (Afshan et al. 2010, 2011a,b). During 
a 2007 survey of the rust flora of Fairy Meadows, four members of Poaceae 
infected with rust fungi were collected. Among these, Puccinia brachypodii var. 
major on Poa attenuata and P. substriata var. indica on Pennisetum orientale 
represent new records for Pakistan, while P brachypodii var. poae-nemoralis on 
Anthoxanthum odoratum and P. poarum on Poa pratensis are additions to the 
rust flora of Fairy Meadows. Anthoxanthum odoratum represents a new host for 
rust fungi from Pakistan. 


Materials & methods 

Freehand sections of infected tissue and spores were mounted in lactophenol and 
gently heated to boiling. The preparations were observed under a NIKON YS 100 
microscope. Drawings of spores and paraphyses were made using a Camera Lucida 
(Ernst Leitz Wetzlar, Germany). Spores were measured using an ocular micrometer. 
At least 25 measurements per fungal structure were taken. Both light microscope and 
scanning electron microscope (SEM) images were obtained of the spores. Collections 


AO8 ... Afshan, Khalid & Niazi 


have been deposited in the herbarium of the Botany Department, at the University of 
the Punjab, Lahore (LAH). 


Enumeration of taxa 


Puccinia brachypodii var. major Cummins & H.C. Greene, Mycologia 58: 711 
(1966) Fics A-B 


SPERMOGONIA and AEcIA unknown. UREDINIA amphigenous, subepidermal, 
light brown, 0.6-0.8 x 1.9-2.0 mm. UREDINIOSPORES globose to subglobose 
or obovoid, (16-)21-24(-29) x (23-)27-35(-39) um (mean 22 x 30 um); 
wall 1.5-2 um thick, light yellow to pale brown, echinulate; germ pores (6-) 


Fics. A-B: Puccinia brachypodii var. major. 
A: Urediniospores and paraphyses. B: Teliospores. Scale bars = 10 um. 


Puccinia species on Poaceae (Pakistan) ... 409 


8-11, scattered, obscure; pedicel hyaline, 6-8 um wide and 32-50 um long. 
PARAPHYSES 70-95 um long, hyaline to pale yellow, capitate, head 15-17 um 
wide; wall = 2 um thick. TELIA amphigenous, sub-epidermal, loculate, with 
stromatic paraphyses, dark brown, 0.4-0.7 x 0.6-0.8 mm. TELIOsPORES 1-2 
celled, oblong to clavate, golden brown to chestnut brown but paler basally, 
smooth; 20-26 x (27—)39-43(-55) um (mean 26 x 34 um); wall 1.5-2 um thick; 
apex truncate or rounded, sometimes conical, 3-5 um thick; germ pore 1 per 
cell, obscure; pedicel short, hyaline, 6-10 x 31-45 um. 

MATERIAL EXAMINED: PAKISTAN, NORTHERN AREAS, Fairy Meadows, at 3036 ma.s.l., 

stages II + III, on Poa attenuata Trin., 12 August, 2007, NSA # 52. (LAH NSA1034). 
ComMENTSs: Puccinia brachypodii var. major is a new record for Pakistan, and 
Poa attenuata is a new host record for this fungus. Previously this rust has been 
reported on Poa horridula and P. candamoana from Peru (Cummins 1971). 

In Pakistan, Puccinia brachypodii var. brachypodii has been reported on 
Brachypodium sp. from KPK and Kaghan Valley (Kakishima et al. 1993b); 
and P. brachypodii var. poae-nemoralis on Agrostis munroana, Poa nemoralis, 
P. pratensis, and P. sterilis from Kaghan Valley, Sharan, Swat, and Azad Jammu 
& Kashmir (Ahmad 1956a; Kakishima et al. 1993a,b; Masood et al. 1995). 


Puccinia substriata var. indica Ramachar & Cummins, Mycopath. Mycol. appl. 25: 
30 (1965) Figs C-D 
SPERMOGONIA and AEcIA unknown. UREDINIAamphigenous, sub-epidermal, 
yellowish-brown to golden-brown, 0.09-0.1 x 0.1-2.0 mm. UREDINIOSPORES 
variable in shape, globose to subglobose or ovoid to ellipsoid, 18-24(-29) x 
21-24(-30) um, pale brown to golden brown, echinulate; wall 1-1.5 um thick; 
germ pores 3-4(-5), equatorials; pedicel hyaline, 4-5 um wide and = 15 um 
long. TeL1a amphigenous, mostly adaxial, sub-epidermal, erumpent, dark 
brown to blackish brown, 0.09-0.5 x 0.2-0.6 mm. TELIOsPORES 1-2-celled, 
oblong to clavate, 17-24(-29) x (29-)34-52(-60) um, cinnamon brown to 
golden brown, paler basally, smooth; wall 2-3 um thick; apex mostly truncate, 
sometimes rounded, 4-9 um thick; germ pores obscure; pedicel hyaline to light 
brown, 6-8 x 8-12 um. 
MATERIAL EXAMINED: PAKISTAN, NORTHERN AREAS, Fairy Meadows, at 3036 m 
a.s.l., stages II + III, on Pennisetum orientale Rich., 12" August, 2007, NSA # 70. (LAH 
NSA1095). 
ComMENTS: Puccinia substriata var. indica is a new record for Pakistan. The 
variety has been reported from India on Pennisetum typhoides (Cummins 1971), 
and Afshan et al. (2008) reported another variety, P. substriata var. insolita (on 
Panicum antidotale) from Munchinabad in Pakistan. Puccinia penniseti-lanati 
(on P lanatum) and Uromyces penniseti (on P americanum from Kaghan 
Valley, Tandojam, and Karachi are two other poaceous rusts known to occur 


410 ... Afshan, Khalid & Niazi 


Fics. C-D: Puccinia substriata var. indica. 
C: Urediniospores. D: 1-2-celled teliospores. Scale bars = 10 um. 


on Pennisetum species in Pakistan (Ahmad 1969; Hasnain et al. 1959; Khan & 
Kamal 1968). 


Puccinia brachypodii var. poae-nemoralis (G.H. Otth) Cummins & H.C. Greene, 
Mycologia 58: 705 (1966) Fics E-F 


SPERMOGONIA and AECIA unknown. UREDINIA on leaves, mostly adaxial, 
yellowish or yellowish brown, 0.09-0.2 x 0.2-0.4 mm. UREDINIOSPORES 
obovoid or ellipsoid to broadly ellipsoid, 19-26 x 23-36 um (23.2 x 28.6 um); 
wall 2-3 um thick, light brown to cinnamon brown, closely echinulate; germ 
pores 5-9, obscure; pedicel hyaline, short, 7-8 x 38-45 um. PARAPHYSES 
cylindric to capitate, hyaline or yellowish, mostly 80-100 um long and 14-18 
um wide, usually geniculata, wall 2-4 um thick throughout or to 6 um thick in 
the head. TeL1a on leaves, mostly abaxial, sub-epidermal, 0.06-0.1 x 0.1-0.2 
mm, blackish, loculate with a few brown paraphyses surrounding the sori. 
TELIOSPORES 1-2 celled (few one-celled spores also observed), oblong to clavate, 
13-24 x 40-59(-65) um (mean 17.7 x 50.6 um), not or slightly constricted at 


Puccinia species on Poaceae (Pakistan) ... 411 


Fics. E-F: Puccinia brachypodii var. poae-nemoralis. 
E: Urediniospores and paraphyses. F: Teliospores. Scale bars = 10 um. 


the septum; wall 1-1.5 um thick, smooth, brown to chestnut brown or paler 
basally; apex 5-9 um thick, truncate or conical; germ pore 1 per cell, upper 
sub-apical, lower at the equator or near the septum; pedicel short, light brown, 
5-6 x 9-15 um, not collapsing, thick walled. 

MATERIAL EXAMINED: PAKISTAN, NORTHERN AREAS, Karimabad, Hunza, at 2438 m 

a.s.l., stages I] + III, on Anthoxanthum odoratum L., 13 August, 2007; Fairy Meadows, 

at 3036 m a.s.l., 12 August, 2007, NSA # Gr 58 (G06). (LAH NSA1035a & 1035b). 
ComMENTs: Puccinia brachypodii var. poae-nemoralis has been reported on 
leaves of Agrostis munroana, Poa nemoralis, P. pratensis, and P. sterilis from 
Kaghan Valley, Sharhan, Swat and Azad Jammu & Kashmir (Ahmad et al. 
1997). 


412 ... Afshan, Khalid & Niazi 


P. brachypodii var. poae-nemoralis is reported for the first time from Fairy 
Meadows and Hunza, Northern Areas of Pakistan; Anthoxanthum odoratum is 
a new host for rust fungi from Pakistan. 


Puccinia poarum P. Nielsen, Bot. Tidsskr., ser. 3, 2: 34 (1877) Fics G-H 

SPERMOGONIA and AEcIA unknown. UREDINIA amphigenous, subepidermal, 
light yellow to light brown, 0.6-0.8 x 1.9-2.0 mm. UREDINIOSPORES globose to 
subglobose or obovoid, 20-26 x 23-30 um (mean 22.92 x 26.71 um); wall 1.5-2 
uum thick, light yellow to pale brown, echinulate; germ pores 4—-5(-8), obscure; 
pedicel hyaline, 5-6 um wide and = 15 um long. PARAPHysEs few, clavate to 
capitate, peripheral, hyaline to pale yellow, head 19-21 um wide, wall = 2 um 
thick, 94-100 um long. TELIA amphigenous, covered by the epidermis, rarely 


Fics. G-H: Puccinia poarum. 
G. Urediniospores and paraphyses. H: Teliospores. Scale bars = 10 um. 


Puccinia species on Poaceae (Pakistan) ... 413 


loculate, dark brown, 0.4-0.7 x 0.6-0.8 mm. TELIOSPORES 1-2 celled, oblong to 
clavate; wall 2-3 um thick, golden brown to chestnut brown but paler basally, 
smooth; 11-19(-22) x 37-58(-67) um (mean 15.7 x 47.9 um); apex truncate 
or conical to obliquely conical, 3-5 um thick; germ pore 1 per cell, obscure; 
pedicel short, brown, 6-7 x 9-11 um. 


MATERIAL EXAMINED: PAKISTAN, NORTHERN AREAS, Fairy Meadows, at 3,036 ma.s.L., 
stages II + III, on Poa pratensis L., 12 August, 2007, NSA # G 371 (LAH NSA1077). 


COMMENTS: Puccinia poarum has been reported on Poa annua from Lahore 
by Ahmad (1956a,b), on Agrostis sp. from Kaghan valley and Azad Jammu & 
Kashmir by Kakishima et al. (1993b). Puccinia poarum is newly recorded from 
Fairy Meadows, Northern areas of Pakistan. 


Acknowledgements 

We sincerely thank Dr. José R. Hernandez, Risk Manager (Plant Pathology 
Regulations, Permits & Manuals, USDA) and Dr. Omar Paino Perdomo (Dominican 
Society of Mycology Santo Domingo, Dominican Republic) for their valuable 
suggestions to improve the manuscript and acting as presubmission reviewers. We are 
highly obliged to the Higher Education Commission (HEC) of Pakistan for providing 
financial support for this research work. 


Literature cited 

Afshan NS, Khalid AN, Javed H. 2008. Further additions to the rust flora of Pakistan. Pakistan 
Journal of Botany 40(3): 1285-1289. 

Afshan NS, Khalid AN, Niazi AR. 2010. Three new species of rust fungi from Pakistan. Mycological 
Progress 9: 485-490. http://dx.doi.org/10.1007/s11557-010-0655-8 

Afshan NS, Khalid AN, Niazi AR, Iqbal SH. 2011a. New records of Uredinales from Fairy Meadows, 
Pakistan. Mycotaxon 115: 203-213. http://dx.doi.org/10.5248/115.203 

Afshan NS, Khalid AN, Iqbal SH, Niazi AR. 2011b. Puccinia species new to Azad Jammu & Kashmir, 
Pakistan. Mycotaxon 116: 175-182. http://dx.doi.org/10.5248/116.175 

Ahmad S. 1956a. Uredinales of West Pakistan. Biologia 2(1): 29-101. 

Ahmad S. 1956b. Fungi of Pakistan. Biological Society of Pakistan, Lahore Monograph 1: 1-126. 

Ahmad S. 1969. Fungi of Pakistan. Biological Society of Pakistan, Lahore, Monograph 5(Suppl. 
1): 110. 

Ahmad §, Iqbal SH, Khalid AN. 1997. Fungi of Pakistan. Nabiza Printing Press, Karachi, Pakistan. 

Cummins GB. 1971. The rust fungi of cereals, grasses and bamboos. Springer Verlag, Berlin- 
Heidelberg—New York. 

Hasnain SZ, Khan A, Zaidi AJ. 1959. Rust and smut of Karachi. Department of Botany, University 
of Karachi, Monograph 2. 36 p. 

Kakishima M, Izumi O, Ono Y. 1993a. Rust fungi (Uredinales) of Pakistan collected in 1991. 
Cryptogamic Flora of Pakistan 2: 169-179. 

Kakishima M, Izumi O, Ono Y. 1993b. Graminicolous rust fungi (Uredinales) from Pakistan. 
Cryptogamic Flora of Pakistan 2: 181-186. 

Khan SA, Kamal M. 1968. The fungi of South West Pakistan. Part 1. Pak. J. Sci. & Ind. Res. 11: 
61-80. 

Masood A, Khalid AN, Iqbal SH. 1995. New records of graminicolous rust fungi (Uredinales) from 
Pakistan. Sci. Int. 7(3): 415-416. 


ISSN (print) 0093-4666 © 2012. Mycotaxon, Ltd. ISSN (online) 2154-8889 


MY COTAXON 


http://dx.doi.org/10.5248/120.415 
Volume 120, pp. 415-422 April-June 2012 


Lectotypification and characterization of the natural phenotype 
of Fusarium bactridioides' 


KEITH A. SEIFERT & TOM GRAFENHAN 


Biodiversity (Mycology & Botany), Agriculture & Agri-Food Canada, 
Ottawa, ON, K1A 0C6 Canada 

Grain Research Laboratory, Canadian Grain Commission, 
Winnipeg, MB, R3C 3G8 Canada 


* CORRESPONDENCE TO: keith. seifert@agr.gc.ca 


ABSTRACT — Specimens of Fusarium bactridioides deposited in the US National Fungus 
Collections and New York Botanical Garden were examined and a lectotype is selected based 
on naturally infected galls of Cronartium conigenum collected in Arizona. This species was 
previously only described from culture, and its natural phenotype is presented and illustrated 
here. Historical experiments involving the attempted use of F. bactridioides as a biocontrol of 
pine blister rusts in Oregon and New Hampshire are reviewed, but the unpublished records 
of the ultimate fate of these experiments could not be located. 


Key worps — Hypocreales, Nectriaceae, anamorph taxonomy, biological control 


Introduction 

Fusarium bactridioides was the last species of the genus to be formally 
described in the august pages of the journal SciENcE. The single page outlined the 
circumstances of the discovery of the fungus by the American botanist Arthur 
Hinckley and forest pathologist L.N. Goodding. The Fusarium “thoroughly 
parasitized” a cone of Pinus leiophylla var. chihuahuana (Chihuahua pine) 
attacked by the cone blister rust, Cronartium conigenum Hedgc. & N.R. Hunt. 
It was originally collected in the remote Chiricahua Mountains in Arizona (ca. 
31°50'N 109°17'W), a so-called ‘sky island’ range of eroded volcanic rhyolite 
rock arising from the surrounding grassland desert. 


' This article, the first of a projected series on the typification and nomenclature of species of 
Fusarium and allied genera, is dedicated to our friend and mentor Emory G. Simmons, whose 
erudite and articulate ‘Alternaria: Themes and Variations, published in this journal, inspired 
this pale imitation. 


416 ... Seifert & Grafenhan 


Following the formal description is a remarkable story of what must be among 
the earliest deliberate releases of one fungus as a possible biological control of 
another. The Cronartium host is one of several rusts infecting Pinus species in 
the southwestern United States and adjacent Mexico, with uredinia and telia 
developing on various Quercus spp. (Cannon 2007). In July 1932, only three 
months after the discovery of the species, conidia of F. bactridioides were sprayed 
onto living C. conigenum cankers on Pinus monticola in Clackamas County 
Oregon. The following July, all the inoculated cankers were dead. Inoculations 
were then attempted on galls of C. harknessii E. Meinecke and C. filamentosum 
Hedge. on P. contorta in two locations in Oregon, where sporulating Fusarium 
colonies were observed on the galls during a survey four months later. Field 
inoculations in four localities in Idaho were reported, but only sketchy details 
were provided of observations of the Fusarium sporulating on the cankers 
afterwards and there are no subsequent literature reports. Unfortunately, we 
were unable to locate any unpublished notes of L.N. Goodding that might give 
more information on the ultimate fate of these inoculations. 

Fusarium bactridioides was classified in Fusarium sect. Discolor by 
Wollenweber (1934), Wollenweber & Reinking (1935), and Gerlach & 
Nirenberg (1982). It was considered a synonym of F. sambucinum Fuckel (Booth 
1971, Nelson et al. 1983) and E bactridioides vaguely fits the broad concept of 
F. sambucinum adopted by these authors. However, the macroconidia lack 
the asymmetrical, ‘dolphin-nose’ apical cell typical of the latter species. Of the 
species segregated from F. sambucinum by Nirenberg (1985), the macroconidia 
of F. bactridioides have some morphological similarity to those of the potato 
pathogen E venenatum Nirenberg, but the latter species lacks microconidia. The 
phylogenetic and taxonomic studies of O’Donnell et al. (1998) and Nirenberg 
& O'Donnell (1998) accept F. bactridioides as a distinct species in the American 
clade of the Fusarium fujikuroi complex. 

Wollenweber (1934) did not illustrate the species with the protologue, 
but later published a drawing as Fusarium autographice delineata no. 1153 
(Wollenweber 1935), which was excerpted in his subsequent monographs. All 
published descriptions and illustrations of this species are probably based on 
one culture, exemplified by the modern species description and microscopic 
photographs of Gerlach & Nirenberg (1982: 267). This in vitro characterization 
is not repeated here, but the natural ‘wild type’ of this species on pine rust galls 
is illustrated and described. No holotype was designated in the protologue, and 
this shortcoming is remedied below by the selection of a lectotype. 


Typification 
The protologue of F. bactridioides explicitly mentions three specimens, the 
first probably the original collection from Arizona, and two from the inoculation 


Fusarium bactridioides lectotypified ... 417 


Fics. 1-2. Fusarium bactridioides, habit photographs. 1. Naturally infected Cronartium conigenum 
gall (BPI 451322). 2. Detail of sporodochia (lectotype, composite photograph assembled with 
CombineZ, Hadley 2006). 


experiments in Oregon. There is no designation of a holotype. The description 
was based on a culture, and it is unlikely that Wollenweber ever saw any of the 
specimens that he listed. 

Eight specimens of E bactridioides are deposited in BPI and one in NY. Apart 
from one specimen collected and identified by J.R. Hansbrough in Waterville, 
New Hampshire (BPI US0451805) after the publication of the protologue, these 
collections are authentic and represent; a) material collected by A. Hinckley 
in the Chiricahua Mountains (NY 00936830 from 1932 on a host identified as 
Cronartium quercuum (Berk.) Miyabe; BPI US0451322 from 1933 on a host 
identified as C. conigenum); b) dried cultures from “cones collected by Arthur, 
Mar. 15, 1934” (BPI US0451803) and four specimens collected in 1933 from 
the inoculated locations in Oregon. It seems reasonable to designate the galls 
of C. quercuum collected by Hinckley in 1932 as the lectotype, despite the 
absence of the variety name for the host. The dates and locality match precisely 
and the identification of the host probably was changed between the times 
of specimen deposit and publication. This is presumably the specimen from 
which the cultures and subsequent specimens from inoculated localities were 
derived. The 1933 specimen in BPI appears also to be a natural infection from 
the type locality and although it predates publication, it is not mentioned in the 
protologue. 

An argument could be made to designate the dried cultures in US0451803 as 
the lectotype, because they probably represent the strain(s), but not the actual 
transfers, that Wollenweber described. Unfortunately, the six dried PDA slants 
have no evident sporodochia or macroconidia, although there are abundant 


418 ... Seifert & Grafenhan 


microconidia in the cottony, white aerial mycelium. The cultures are heavily 
contaminated with coccoid bacteria. The date on the package containing the 
dried cultures is “Mar. 15, 1934”, a scant three months before Wollenweber’s 
description was published. The notes inside this packet suggest that the 1934 
date is actually when the preserved cultures were dried. Only one strain of this 
fungus now exists, i.e. Wollenweber 4748 > CBS 177.35 (> BBA 63602, CBS 
> NRRL 22201, NRRL > DAOM 225115, NRRL > CBS 100057). The data for 
CBS 100057 record P. leiophylla as the plant host and the location as Arizona, 
suggesting that this strain, which was regarded as an ‘ex-type culture’ by 
Gerlach & Nirenberg (1982) and Nirenberg & O'Donnell (1998), was derived 
from material from the Chiricahua Mountains. The protologue is ambiguous 
about how many strains were originally isolated and how many were sent to 
Wollenweber by his American colleagues. One of these strains was illustrated as 
Fusarium autographice delineata no. 1153, and we must assume that the culture 
now represented by CBS 100057 is the same strain. We consider this to be the 
ex-lectotype strain, and see no need to formalize this by epitypification. 

For formal typification, we prefer to emphasize the concept of the species as 
a parasite of Cronartium, and designate the lectotype accordingly below. 


Taxonomy 


Fusarium bactridioides Wollenw., Science 79: 572. 1934. FIGS 1-5 
MycoBank MB 258078 


Type: On Cronartium quercuum on Pinus chichuahuana (sic), USA, Arizona, Chiricahua 
Mt., Cave Creek, HI.1932, Arthur Hinckley (lectotype designated here, NY 00936830). 


Sporodochia are erumpent from host tissue and visible as Orange-White 
(5A2, Kornerup & Wanscher 1978) to Light Orange (5A5) masses of conidial 
slime. On the naturally infected specimens, they are irregular in outline and 
form lesions up to 1 cm x 5 mm on the galls. On inoculated specimens, the 
lesions are scattered and smaller, about 200-1000 um diam. The stroma is 
poorly developed, about 100 um thick, and in optical section appears as a 
textura angularis of thin- to slightly thick-walled cells about 3.5-7 um wide, but 
its hyphal character is evident with changes of focus. A hymenium-like layer of 
conidiophores and phialides arises from the stroma, with the conidiophores 
more or less biverticillate, but with phialides also often arising at the first level 
of branching. Metulae are cylindrical, doliiform to slightly clavate, 8.5-11.5 x 
3.5-7 um at the broadest part. Phialides are 9.5-15 x 3-5 um at the broadest 
part, narrowly doliiform, sometimes with a central constriction, in terminal 
pairs or whorls of 3, or arising singly, in pairs or in whorls with metulae, with 
a flared collarette about 1-1.5 um long and periclinal thickening sometimes 
visible; conidiogenous aperture about 2 um wide. 


Fusarium bactridioides lectotypified ... 419 


+ - : 1 ™ _ 4 
Fics 3-4. Microphotographs of Fusarium bactridioides, BPI 451804. 3. Macroconidia. (composite 
image). 4. Hand section of the sporodochial stroma, showing conidiophores and phialides. 
Scale bars = 10 um. 


There is no obvious distinct difference between ‘microconidial’ and 
« *4°_,p . ‘i . 
macroconidial’ sporodochia and there is more or less a continuum between 
the two conidial types. Few macroconidia occur on the naturally infected 


420 ... Seifert & Grafenhan 


specimens, but they are more abundant on the lectotype and inoculated 
specimens. Macroconidia are 3-6 septate; 3-septate predominate and are 
30.5-45.5 x 4.5-6 um (mean + SE = 38.3 + 0.8 x 5.3 + 0.1, n = 20), I/w = 5.5-9; 
4-septate 27-36.5 x 5-6 um (mean + SE = 31.9+0.8 x 5.4+0.1,n= 10), l/w=5-7; 
5-septate 29-39.5 x 5.5-6.5 um (mean + SE = 34.6 + 0.9 x 5.8 + 0.1, n = 13), 
I/w = 5-7; 6-septate 35.5-41 x 5-6 um (n = 3), I/w = 6-7. In side view, the 
ventral surface is more or less straight or gently curved, and the dorsal surface 
is moderately curved, with the walls more or less parallel in the central two cells 
of the conidia, with the widest point near the middle or above the middle. The 
apical cell is bluntly rounded, and roughly the same length as the penultimate 
cell. The basal cell is tapered more acutely than apical cell; the base is rounded, 
flat, or has a slight indentation on the dorsal side or central papilla, indicating a 
foot cell. In front view, the macroconidia appear somewhat clavate. 4-6 septate 
macroconidia tend to have unequal lengths of cells, with the additional septa 
dividing one but not all of the original four cells. Microconidia are abundant 
in the sporodochia, are 0-3 septate, and vary in shape and size from small, 
ellipsoidal, oblong-ellipsoidal or allantoid cells that are obviously microconidia 
to fusiform to clavate, septate spores that intergrade with macroconidia. 
Aseptate conidia 6-18 x 3.5-6.5 um (mean + SE = 11.8 + 0.6 x 4.9 + 0.1, n = 25), 
I/w = 1.5-4. 1-septate conidia 9.5-19.5 x 4-6.5 um (mean + SE = 14.9 + 0.5 
x 5.1 + 0.1, n = 25), l/w = 2-4. 2-septate conidia are infrequent and 15-24 x 
5-6.5 uum. 3-septate microconidia also occur and can be distinguished from 
macroconidia by their shorter length and their more clavate shape; they are 
16-31 x 5-7 um (mean + SE = 24.0 + 0.7 x 6.1 + 0.1, n = 25), I/w = 3-5. In 
general, the bases of microconidia are conical, symmetrical or asymmetrical, 
with a flat secession scar or relatively conspicuous papilla; the apical cell or part 
of the conidium is rounded. 

ADDITIONAL SPECIMENS EXAMINED: USA, Arizona, Chiricahua Mt. (as Chricicalma), 

Cave Creek, on Cronartium conigenum on Pinus leiophylla, 25.V1I1.1933, Arthur Hinckley 

(BPI US0451322); Oregon, Mt. Hood National Forest, On Cronartium harknessii on 

Pinus contorta, 8.V1I.1933, L.N. Goodding (BPI US0451323); on Cronartium ribicola, 

24.X.1933, L.N. Goodding (BPI US0451325); Eagle Creek, on Cronartium ribicola on 

Pinus monticola, 24.X.1933, L.N. Goodding (BPI US0451326); six dried slant cultures on 

PDA from “cones collected by Arthur” (BPI US0451803); Hood River Co., Eagle Creek, 

on Cronartium ribicola on Pinus sp., 24.X.1933, L.N. Goodding (BPI US0451804); 


New Hampshire, Waterville, on Pinus strobus, 1.[X.1935, J.R. Hansbrough (BPI 
US0451805). 


Discussion 

The micromorphology of F. bactridioides reported here from natural 
material is comparable to that reported from cultures of this species by 
Wollenweber (1934) and Gerlach & Nirenberg (1982). The main distinction 
is that on the natural substrate the microconidia are produced from the 


Fusarium bactridioides lectotypified ... 421 


Fic. 5. Microphotograph of microconidia of Fusarium bactridioides, lectotype, NY 00936830 
(composite image). Scale bar = 10 um. 


same sporodochia as the macroconidia, whereas in culture microconidia are 
produced independently. In nature, there is a continuum of shape and size 
between macro- and microconidia, but in culture the two kinds seem to be 
morphologically distinct. Gerlach & Nirenberg (1982) reported macroconidia 
with up to 11 septa in culture, with 3-5 septa being the typical condition. On 
the specimens, the macroconidia with three septa predominated, with a small 
number of 4-6 septate macroconidia seen on some specimens. Morphologically, 
Fusarium bactridioides has more robust macroconidia than is typical for the F. 
fujikuroi complex, and the 0-1 septate oval macroconidia are produced on the 
agar surface in vitro, rather than in the aerial mycelium as they are in related 
species. The mycoparasitic habit is also unusual in this group. 

To our knowledge, Fusarium bactridioides has not been collected after 
1935 and no natural infections have been reported outside of Arizona. The 
Hansbrough specimens (USO451805) collected in 1935 are the result of 
inoculation experiments in New Hampshire in 1934, about which apparently 
nothing was ever published. We were unsuccessful in locating the typewritten 
report by Hansbrough cited in a note included with the specimens. Whether 
EF. bactridioides is indigenous and perhaps restricted to the remote Chiricahua 
Mountains, and whether the inoculations of blister rust cankers in Oregon, 


422 ... Seifert & Grafenhan 


Idaho and New Hampshire still persist, seem questions worthy of investigation. 
Such inoculation experiments would not easily be conducted today, with the 
need for risk assessments and legal permits from regulatory agencies. Fusarium 
bactridioides may provide a unique opportunity to search for the lingering 
fingerprints of innovative scientific activities of a more innocent time. 


Acknowledgments 

We are grateful to Helgard Nirenberg and Walter Gams for their insights into the 
practices of H.W. Wollenweber in Berlin, the curators of BPI and NY for the kind 
loans of specimens discussed in this article, and Drs T. Aoki and B. Summerell for pre- 
submission reviews. We appreciate the efforts of staff at the herbarium (S. Hunkins) 
and library (M. Carpenter) of the University of Arizona to find archival material of L.N. 
Goodding, and staff at the Connecticut Agricultural Experiment Station (D.W. Li, S. 
Anagnostakis, V. Bomba-Lewandoski, W. Elmer) and USDA Forestry Lab in Hamden, 
CT (M. Keena) for searching for archives of J.R. Hansbrough. 


Literature cited 

Booth C. 1971. The genus Fusarium. Commonwealth Mycological Institute, Kew. 

Cannon PE. 2007. Cronartium conigenum. IMI descriptions of fungi and bacteria No. 1722. 4 p. 

Gerlach W, Nirenberg HI. 1982. The genus Fusarium - a pictorial atlas. Mitt. Biol. Bundesanst. 
Ld.- u. Forstw. 209: 1-406. 

Hadley A. 2006. CombineZ, version 5. Published by the author. www.hadleyweb.pwp 

Kornerup A, Wanscher JH. 1978. Methuen handbook of color, 3rd ed. Denmark, Sankt Jorgen 
Tryk. 243 p. 

Nelson PE, Toussoun TA, Marasas WFO. 1983. Fusarium species, an illustrated manual for 
identification. Pennsylvania State Univ. Press, University Park, London. 193 p. 

Nirenberg HI, O'Donnell K. 1998. New Fusarium species and combinations within the Gibberella 
fujikuroi species complex. Mycologia 90: 434-458. http://dx.doi.org/10.2307/3761403 

O’Donnell K, Cigelnik E, Nirenberg HI. 1998. Molecular systematics and phylogeography of the 
Gibberella fujikuroi species complex. Mycologia 90: 465-493. 
http://dx.doi.org/10.2307/3761407 

Wollenweber HW. 1934. Fusarium bactridioides sp. nov., associated with Cronartium. Science 79: 
572. http://dx.doi.org/10.1126/science.79.2060.572 

Wollenweber HW. 1935. Fusaria autographice delineata [4]: nos 1101-1200. Self published, Berlin. 
1200 p. 

Wollenweber HW. 1943. Fusarium—Monographie IJ. Fungi parasitici et saprophytici. ZenBl. Bakt,, 
Abt. 2, 106: 171-202. 

Wollenweber HW, Reinking OA. 1935. Die Fusarien, ihre Beschreibung, Schadwirkung und 
Bekampfung. Paul Parey, Berlin. 355 p. 


ISSN (print) 0093-4666 © 2012. Mycotaxon, Ltd. ISSN (online) 2154-8889 


MY COTAXON 


http://dx.doi.org/10.5248/120.423 
Volume 120, pp. 423-426 April-June 2012 


Lectotypification of Crepidotus variabilis var. subsphaerosporus 


SONA JANCOVICOVA' & SHAUN R. PENNYCOOK?’ 


' Comenius University in Bratislava, Faculty of Natural Sciences, 
Department of Botany, Révovd 39, 811 02 Bratislava, Slovakia 
? Manaaki Whenua Landcare Research, 
Private Bag 92 170, Auckland 1142, New Zealand 


CORRESPONDENCE TO: ' jancovicova@fns.uniba.sk & * PennycookS@LandcareResearch.co.nz 


ABSTRACT — An original watercolour painting by J.E. Lange is designated as the lectotype 
of Crepidotus variabilis var. subsphaerosporus. This painting was cited in two descriptions 
published invalidly by Lange prior to his validation of the name; it therefore constitutes part 
of the original material on which he based his description. As a lectotype, it supersedes the 
neotype previously designated by Senn-Irlet, which is here designated as epitype. 


Key worps — Afbildninger af Danmarks Agaricaceer, Basidiomycota, Crepidotus cesatii var. 
subsphaerosporus, Crepidotus kubickae, Flora Agaricina Danica 


Introduction 

The name Crepidotus variabilis var. subsphaerosporus was first proposed by 
J.E. Lange (1938: 52, as ‘subsphaerospora’) as a nomen non rite publicatum, 
lacking a Latin description (McNeill et al. 2006: Art. 36.1). No type specimen 
was designated; according to M. Lange (1969: 131), J.E. Lange “never tried to 
make up a set of herbarium specimens.’ However, an illustration was cited 
(as “D.A. pl. 533”), together with details of collection locality, habitat, and 
date. The abbreviation “D.A.” refers to the extensive series of watercolours 
painted by J.E. Lange during the years 1893-1910 and compiled under the 
title “Afbildninger af Danmarks Agaricaceer” (J.E. Lange 1914: 1; M. Lange 
1969: 122-123). The original paintings (initially held by J.E. Lange) are now 
conserved in the Herbarium, Natural History Museum of Denmark, University 
of Copenhagen (C), and are reproduced on the Flora Agaricina Danica 
website (http://130.225.211.158/agaricina/flagar-search.htm; H. Knudsen, 
pers. comm.). Subsequently, J.E. Lange painted a duplicate set for the Museum 
on better quality paper (M. Lange 1969: 123); this set is now held in the 
private collection of his granddaughter, Dr. Lene Lange, Aalborg University 
(H. Knudsen, pers. comm.). 


424 ... Jan¢éovitova & Pennycook 


J.E. Lange (1939: 46, pl. 133 fig. E) republished the description with minor 
changes accompanied by a printed reproduction of the original painting (with 
some repositioning of component elements), but the name remained invalid 
for lack of a Latin description. 

J.E. Lange (1940: IV) finally validated the name in a set of Latin diagnoses 
of names invalidly published in “Flora Agaricina Danica.” Although there is no 
direct reference to the earlier publication pages, the trinomial is marked with 
an asterisk, indicating that the name had originally been published in “Studies 
in the Agarics of Denmark” during the period “1935-40” (J.E. Lange 1940: IJ), 
i.e., in the series of papers that included J.E. Lange (1938). 

When Senn-Irlet (1995: 53) recombined the name as Crepidotus cesatii var. 
subsphaerosporus (J.E. Lange) Senn-Irlet, she designated a Swiss collection as 
neotype. However, this designation ignored the availability of original material 
(the watercolour painting) that should have been selected as lectotype (McNeill 
et al. 2006: Art. 9.10, 9.11). 

Here we designate the original watercolour painting as lectotype, and the 
superseded Swiss “neotype” as epitype. 


Taxonomy and typification 

The name Crepidotus variabilis var. subsphaerosporus has priority at variety 
rank (e.g., as treated in Senn-Irlet 1995). Ripkova (2009: 272) summarized the 
nomenclatural history of the taxon under Crepidotus kubickae, the synonym 
that has priority at species rank. Two widely cited purported recombinations of 
J.E. Lange's basionym at species rank were not validly published (McNeill et al. 
2006: Art. 33.4): “Crepidotus subsphaerosporus’ (J.E. Lange) Kuhner & Romagn. 
(Kihner & Romagnesi 1953: 76) lacked a full and direct basionym reference, 
and “Crepidotus subsphaerosporus” (J.E. Lange) Kithner & Romagn. ex Hesler & 
A.H. Sm. (Hesler & Smith 1965: 121) gave a full and direct reference to the 1938 
nomen non rite publicatum, a non-correctable nomenclatural error (McNeill 
et al. 2006: Art. 33.7). 

J.E. Lange preserved no herbarium specimens other than an occasional 
spore print. There are no J.E. Lange collections or spore prints of C. variabilis 
var. subsphaerosporus at C; the watercolour painting, conserved at C (viewable 
on http://130.225.211.158/agaricina/flagar-search.htm under “Crepidotus sub- 
sphaerosporus’), is apparently the only extant original material. (J.E. Lange's 
duplicate painting is extant, but it is debatable whether this can be considered 
to be “original material.”) The original painting is on a 26.5 x 17.7 cm sheet of 
thick, cardboard-like, aquarelle paper, which has now yellowed considerably. 
The images comprise a drawing of two basidiospores, a painted representation of 
the spore print colour (easily overlooked because of its resemblance to adjacent 
blotches of foxing discoloration), and three habit paintings of the basidiomata. 


Crepidotus variabilis var. subsphaerosporus, lecto- & epitypified ... 425 


(The three habit paintings and the basidiospore drawing, but not the spore print 
colour representation, are also reproduced in J.E. Lange 1939: pl. 133 fig. E.) 
The original painting bears various handwritten ink and pencil annotations in 
Danish: in the upper right corner “Tavle 533”; below the basidiospore drawing 
“sp x 1000”; and along the lower margin “Crepidotus sessilis (Bull.) Schroet. 
f. subsphaerospora. Sporestov blegt okkerbrunt. Sp spheerisk-ellips, 6 x 4 %. 
Heesbjerg, pa pinde af eg o.a. lovtreer. Okt. 1901” Hzesbjerg is an old spelling 
for the modern Hesbjerg, the name of a number of localities on Funen, the 
Danish island where J.E. Lange lived and did most of his collecting; the most 
likely collection locality is at 55°22'N 10°13’E, the only forested Hesbjerg on 
Funen (H. Knudsen, pers. comm.). 

We propose the original painting as lectotype of C. variabilis var. subsphaero- 
sporus. We also propose Senn-Irlet’s superseded “neotype’” as epitype, providing 
micromorphological data to supplement the macromorphology illustrated in 
the lectotype. 


Crepidotus kubickae Pilat, Stud. Bot. Cechoslov. 10: 150, 1949, as ‘Kubickae’. 


TyPE (Pilat 1949, Senn-Irlet 1992, Ripkova 2009): CZECH REPUBLIC. Central Bohemia: 
“Poricko nad Sazavou, ad terram inter muscis, 29.V.1949, legit J. Kubicka, det. Pilat, 
Typus.” (PRM 665190, holotype). 
= Crepidotus variabilis var. subsphaerosporus J.E. Lange, Fl. Agaric Danic. 5: IV, 1940. 

Types: DENMARK. Funen: Hesbjerg, “Crepidotus sessilis (Bull.) Schroet. f. 
subsphaerospora. Sporeprint pale ochre brown. Sp sphaerical-ellipsoid, 6 x 4%. Heesbjerg, 
on sticks of Quercus and other deciduous trees. Oct. 1901.” [in Danish]. (C, Danmarks 
Agaricaceer Tavle 533, original watercolour painting by J.E. Lange, lectotype designated 
here). SWITZERLAND. Bern: Rothenbach, Schineggschwand am Schallberg, alt. 1000 
m, Abieti-Fagetum, on fallen Picea twigs, 11 Oct. 1989, Senn-Irlet 89/240 (G, epitype 
designated here). 


Acknowledgments 

We thank Henning Knudsen (the Herbarium, Natural History Museum, University 
of Copenhagen), without whose willingness to answer numerous questions concerning 
J.E. Lange’s paintings and their curation this paper could not have been written, and 
Henning Knudsen and Scott Redhead (Systematic Mycology and Botany, Agriculture 
and Agri-Food Canada, Ottawa) for refereeing the manuscript. 


Literature cited 

Hesler LR, Smith AH. 1965. North American species of Crepidotus. Hafner Publishing Company, 
New York. 168 p. 

Kiihner R, Romagnesi H. 1953. Flore analytique des champignons supérieurs (agarics, bolets, 
chanterelles). Masson et Cie, Paris. 556 p. 


Lange JE. 1914. Studies in the agarics of Denmark. I. General Introduction. The genus Mycena. 
Dansk Botanisk Arkiv 1(5): 1-40. 


426 ... Jan¢éoviéova & Pennycook 


Lange JE. 1938. Studies in the agarics of Denmark. Part XII. Hebeloma, Naucoria, Tubaria, 
Galera, Bolbitius, Pluteolus, Crepidotus, Pseudopaxillus, Paxillus. Additional descriptions and 
supplementary notes to part I-XI. Dansk Botanisk Arkiv 9(6): 1-104. 

Lange JE. 1939. Flora Agaricina Danica, Vol. 4. The Society for the Advancement of Mycology in 
Denmark and the Danish Botanical Society, Copenhagen. pp. 1-119, pl. 121-160. 

Lange JE. 1940. Flora Agaricina Danica, Vol. 5. The Society for the Advancement of Mycology in 
Denmark and the Danish Botanical Society, Copenhagen. pp. 1-103 + I-XXIV, pl. 161-200. 

Lange M. 1969. Jakob E. Lange and the creation of “Flora Agaricina Danica.’ Friesia 9(1-2): 
121-132. 

McNeill J, Barrie FR, Burdet HM, Demoulin V, Hawksworth DL, Marhold K, Nicolson DH, 
Prado J, Silva PC, Skog JE, Wiersema JH, Turland NJ. 2006. International Code of Botanical 
Nomenclature (Vienna Code) adopted by the Seventeenth International Botanical Congress, 
Vienna, Austria, July 2005. Regnum Vegetabile 146. 568 p. 

Pilat A. 1949. Ad monographiam Crepidotorum europaeorum. Supplementum I. Studia Botanica 
Cechoslovaca 10(4): 149-154. 

Ripkova S. 2009. Crepidotus kubickae - a forgotten name. Mycotaxon 110: 271-281. 
http://dx.doi.org/10.5248/110.271 

Senn-Irlet B. 1992. Type studies in Crepidotus — I. Persoonia 14(4): 615-623. 

Senn-Irlet B. 1995. The genus Crepidotus (Fr.) Staude in Europe. Persoonia 16(1): 1-80. 


ISSN (print) 0093-4666 © 2012. Mycotaxon, Ltd. ISSN (online) 2154-8889 


MY COTAXON 


http://dx.doi.org/10.5248/120.427 
Volume 120, pp. 427-435 April-June 2012 


A new species of Conidiobolus (Ancylistaceae) 
from Anhui, China 


YONG Nig?, Cul-ZHu Yu', XIAO- YONG Liu?’ & Bo HUANG" 


‘Anhui Provincial Key Laboratory of Microbial Control, Anhui Agricultural University, 
West Changjiang Road 130, Hefei, Anhui 230036, China 

State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, 
Beijing 100101, China 

* CORRESPONDENCE TO: bhuang@ahau.edu.cn, liuxiaoyong@im.ac.cn 


ABSTRACT —Conidiobolus sinensis was isolated from plant detritus in Huoshan, Anhui 
Province, eastern China. It produces primary conidiophores from cushion mycelium, 
which is distinct from all other species in the genus except C. stromoideus and C. lichenicola. 
Morphologically C. sinensis differs from C. stromoideus in the shape of the mycelia at the 
colony edge and conidiophore length and from C. lichenicola by colony color and mycelial 
form. A phylogram based on partial 28S rDNA and EF-1a sequences from 14 Conidiobolus 
species shows C. sinensis most closely related to C. stromoideus, forming a clade of sister 
taxa with a 100% bootstrap. DNA similarity levels between these two species were 94% (28S 
rDNA) and 96% (EF-1a). Based on the morphological and molecular evidence, C. sinensis is 
considered a new species. 

Key worps —Entomophthorales, hyphal knots, taxonomy 


Introduction 

Species belonging to Conidiobolus can be easily isolated from soil, decaying 
leaf litter, rotten vegetables and some dead insects, although the type of the 
genus, C. utriculosus Bref., was first isolated from the decaying fleshy fruitbodies 
of Exidia and Hirneola. The genus is diagnosed by (i) nuclei that do not stain in 
aceto-orcein and lacking obviously granular contents, (ii) simple conidiophores, 
(iii) globose to pyriform multinucleate conidia, (iv) resting spores formed in the 
axis of hypha (mostly as zygospores), and (v) walled vegetative cells (Humber 
1997). After Huang et al. (2007) recognized 30 species within Conidiobolus, only 
two additional species — C. margaritatus (Huang et al. 2007), C. thermophilus 
(Waingankar et al. 2008) — have been added. 

While many phylogenetic studies of entomogenous fungi have been 
conducted in recent years, there is little information regarding the phylogeny 


A428 ... Nie & al. 


and molecular taxonomy of Conidiobolus. The limited SSU analysis of Jensen 
et al. (1998) suggested that Conidiobolus may be polyphyletic. Vilela et al. 
(2010) were the first to detail the taxonomic and phylogenetic features of three 
pathogenic Conidiobolus: C. coronatus (Costantin) A. Batko, C. lamprauges 
Drechsler, and C. incongruus Drechsler. 

Anhui Province is located at 29°04'-34°06'N 114°09'-119°06’E, eastern 
China, and most parts of the province have subtropical vegetation, but some 
temperate vegetation occurs in mountains. Recently, several new fungal species 
on decaying wood have been reported from the area (Dai 2010; Cui et al. 2011). 
In the course of studies on Conidiobolus species from China, one strain with 
cushion mycelia was isolated from decaying plant material in Anhui and is 
described in this report as a new species, based on both morphological and 
molecular data. 


Materials & methods 


Morphological studies 

Plant detritus was sampled on 17 June 2010 near a reservoir in Huoshan, Anhui, 
China. The sample was screened for saprotrophic Conidiobolus by canopying moistened 
detritus on agar plates following Drechsler (1952) and King (1976a) to exploit the 
forcible discharge of Conidiobolus conidia. These isolation plates were incubated at 21°C 
and examined daily for one week. Once Conidiobolus cultures were detected on the PDA 
canopy, they were transferred to new PDA plates for purification and morphological 
study. The measurements of different fungal structures followed King (1976a). 


Molecular studies 

The Conidiobolus strains used in the molecular study are shown in TABLE 1. Ten 
ex-type strains of Conidiobolus spp. were purchased from American Type Culture 
Collection (ATCC; Manassas, VA, USA), and the remaining Conidiobolus strains were 
obtained from the Research Center for Entomogenous Fungi (RCEF; Anhui Agricultural 
University, Hefei, China). Genomic DNA was extracted using the CTAB method (Yi et 
al. 2003). The extracted DNA was stored in 50-100 uL of HPLC-H,O at -20°C, and 
was diluted 10-fold with HPLC-H,O for use in PCR reactions. Regions of two genes 
were amplified by PCR: 1) nuclear ribosomal large subunit (LSU rDNA) by primers 
LROR and LRS (Vilgalys & Hester 1990) and 2) elongation factor 1-alpha (EF-1a) by 
primers EF983 and EF1aZ-1R (http://www.aftol.org/primers.php). All procedures used 
in this study for LSU amplification have been described previously (Liu et al. 2005). The 
PCR reaction mixture for amplifying EF-1a contained 200 uM each dNTP, 1x Mg-free 
buffer, 2.5 mM MgCl, 0.5 uM each primers, 1 ng/uL genomic DNA, and 0.04 Unit/L 
Taq polymerase. The cycle program included initial denaturation at 100°C for 5 min 
followed by 95°C for 5 min (during which time Taq polymerase was added to each tube), 
34 cycles of 94°C for 1 min, 55°C for 2 min, and 72°C for 2 min, and a final extension 
at 72°C for 10 min. The nucleotide sequences of the PCR products were determined on 
both strands by using dideoxy-nucleotide chain termination on an ABI 3700 automated 
sequencer at Shanghai Genecore Biotechnologies Company. Sequence data of the 19 


Conidiobolus sinensis sp. nov. (China) ... 429 


TABLE 1. Conidiobolus and Entomophthora cultures and sequences used in 
phylogenetic analyses.* 


FUNGAL TAXON STRAIN # 28S RDNA EF-1a 
C. chlamydosporus Drechsler ATCC12242 (T) JF816212 JF816234 
C. denaeosporus Drechsler ATCC12940 (T) JF816215 JF816228 
C. firmipilleus Drechsler RCEF4429 JF816222 JF816237 
C. gonimodes Drechsler ATCC14445 (T) JF816221 JF816226 
C. coronatus RCEF5598 JQO004791 JQ004795 
RCEF5599 JQ004792 JQ004796 
RCEF5600 JQ004793 JQ004797 
RCEF5601 JQ004794 JQ004798 
AFTOL-ID137 AY546691 DQ275337 
C. heterosporus Drechsler RCEF4430 JF816225 JF816239 
C. humicola M.C. Sriniv. & Thirum. ATCC28849 (T) JF816220 JF816231 
C. lichenicola ATCC16200 (T) JF816216 JF816232 
C. lobatus M.C. Sriniv. & Thirum. ATCC18153 (T) JF816218 JF816233 
C. nodosus M.C. Sriniv. & Thirum. ATCC16577 (T) JF816217 JF816235 
C. polytocus Drechsler ATCC12244 (T) JF816213 JF816227 
C. stromoideus ATCC15430 (T) JF816219 JF816229 
C. sinensis RCEF4952 (T) JF816224 JF816238 
C. thromboides Drechsler ATCC12587 (T) JF816214 JF816230 
RCEF4492 JF816223 JF8 16236 
E. muscae (Cohn) Fresen. ARSEF3074 DQ273772 DQ275343 


* The fungal taxonomy follows that of King (1976a, b, 1977). ARSEF = ARS Entomopathogenic 
Fungus Collection (Ithaca, USA). arcc = American Type Culture Collection (Manassas, 
USA). RCEF = Research Center for Entomogenous Fungi (Hefei, China). AFTOL-ID = 
Assembling the Fungal Tree of Life Identity. T = ex type. 


strains of Conidiobolus have been deposited in the GenBank database under the access 
numbers shown in TABLE 1. 

Sequences were aligned with Clustal X (Thompson et al. 1997). The combined data 
of the two loci, partial 28S rDNA and EF-1a, were analyzed with Maximum Parsimony 
(MP) in PAUP* 4.0b10 (Swofford 2003), by using 1000 replicates of heuristic search of 
random sequence additions, branch swapping algorithm by tree bisection-reconnection 
(TBR) and MULTrees in effect. Gaps were treated as missing data and all characters were 
equally weighted. Branch support was estimated by 1000 bootstraps of 10 replicates of 
heuristic search with the same options as the parsimony search (Felsenstein 1985). The 
alignments were fed to DNAMAN software package (Version 5.2.2, Lynnon Biosoft, 
Canada) for calculating genetic similarities. 


Results 

The combined alignment of partial 28S rDNA and the EF-1a dataset was 
1468 bp in length, including 981bp from the LROR/LR5 region of 28S rDNA 
and 487 bp from the EF983/EF1aZ-1R region of EF-1la. 108 sites in 28S rDNA 
and 24 sites in EF-la with ambiguous alignment were excluded from the 
analysis and the final alignment contained 643 parsimony-informative sites. 
Maximum parsimony analysis of 20-taxon dataset resulted in a single tree 
(TL = 1942, CI = 0.6130, RI = 0.7826, HI = 0.3553) shown in PLaTE 1, and 


430 ... Nie & al. 


: €. coronatus RCEF5598 
€. coronatus RCEF5599 
"'F ¢. coronatus RCEFS6O0 
C. coronaius AF TOL-137 
€. coronaius RCEF5601 


99 
€. lichenicola ATCC16200 


2 C. gonimodes ATCC 14445 
7 C. polytocus ATCC12244 
100) C. chlampdosporus ATCC12242 
94 a C. fiemipilleus RCEF4429 
C. Augnicolus ATCC28849 
% C. nodosus ATCC16577 
ig) © Sromoideus —ATCC15430 
C. sinensis. RCEF4952 


100 
jo0( ©. Herammboides ATCC12587 
C. thromboides RCEF4492 
€. denaeosporus ATCC12940 
8 C. heterosporus. RCEF4430 
C. dobaius ATCC18153 
Entomophihora muscae ARSEF3074 


10 changes 


PraTE 1. The single most parsimonious tree (TL = 1942, CI = 0.6130, RI = 0.7826, HI = 0.3553) 
showing phylogenetic relationships among species of Conidiobolus inferred from a combined 
dataset of partial 28S rDNA and EF-la sequences. Bootstrap values =>50 % are labeled above 
relevant branches. Entomophthora muscae served as the outgroup. The bar at the lower left corner 


represents 10 changes. 


Conidiobolus sinensis sp. nov. (China) ... 431 


TABLE 2. Similarities of partial 28S rDNA and EF-1a sequences 
from Conidiobolus strains.* 


% SIMILARITY 


SPECIES— STRAIN sss OO eae. 
1 2 3 4 5 6 7 8 9 10 11 12 13 


1 C. chlamydosporus 99 85 85 67 67 67 65 85 85 85 85 84 


—ATCC12242 
Po ecinipilletts 98 8 85 68 68 68 66 85 85 85 85 85 
— RCEF4429 
3 C. gonimodes 

91 91 97 68 68 66 66 9% 9% %% 9% 96 
—ATCC14445 
SIE CON 90 89 93 68 68 66 66 % 9% 9% £96 9% 
—ATCC16200 
SG faromboiges Feet G34 Fe 76 99 88 86 67 68 68 68 67 
—ATCC12587 
ae ds ats 73 73 75 76 99 88 86 67 68 68 68 66 
— RCEF4492 
Te TMI EUs 7595 75y 77 1 9D 94 65 66 66 65 65 
—ATCC15430 
Ars de 73 73 74 75 91 91 96 65 65 65 65 65 
—RCEF4952 
P CacerenHts 92 92 94 95 76 76 77 75 99 99 98 99 
—RCEF5598 
BOR conan 92 91 94 95 76 76 77 75 99 99 98 99 
—RCEF5599 
pera 91 91 94 9% 76 76 77 «+75 99 99 99 99 
—RCEF5600 
inp giherecs 91 91 93 95 76 76 77 75 98 98 99 98 


—RCEF5601 


13 C. coronatus 
—AFTOL-ID137 


91 91 94 96 76 76 $77 #7 99 99 100 99 


* Data refer to the overall similarities of the partial 288 rDNA (above the diagonal) and 
EF-1a sequences (below the diagonal) 


the DNA similarities among 13 representing strains are listed in TABLE 2. The 
phylogenetic tree shows that the isolate RCEF4952 clustered with the ex- 
type strain of Conidiobolus stromoideus with 100% bootstrap support (PLATE 
1), but the similarities between C. stromoideus and C. sinensis were only 94% 
(28S rDNA) and 96% (EF-1a) (TABLE 2). Higher intraspecific DNA similarities 
were measured from partial 28S rDNA and EF-1a sequence. For example, the 
ranges of DNA similarities within C. coronatus have been found to be 98-99% 
(28S rDNA) and 98-100% (EF-1a), and those within C. thromboides were 99% 
for both genes. On the other hand, the Conidiobolus partial 28S rDNA and EF- 
la showed high genetic divergence among species. The highest similarity of 
partial 28S rDNA (97%) was recorded between C. gonimodes and C. lichenicola, 
and the lowest (65%) between C. coronatus and C. stromoideus. The range of 
similarities in EF-la among species was 73-96%. Although the similarities 
between C. chlamydosporus and C. firmipilleus were 99% (28S rDNA) and 98% 
(EF-1la), C. chlamydosporus was placed in synonymy with C. firmipilleus in 
King’s classification (King 1977). 


432 ... Nie & al. 
Taxonomy 


Conidiobolus sinensis Y. Nie, X.Y. Liu & B. Huang, sp. nov. PLATES 2-3 
MycoBAnk MB563665 
Differs from Conidiobolus stromoideus by its much longer conidiophores and rarely 
branching mycelia at the colony edge. 
Type: China, Anhui Province, Huoshan County, isolated from leaf litter, 17 June 2010, 
[Yong Nie] (Holotype, RCEF4952; GenBank JF816224, jF816238). 


EryMo_oey: sinensis (Lat.) = China, referring to the geographic origin of the strain. 


Colonies grown on PDA for 3 days at 21°C, white, reaching ca 21 mm diameter. 
Numerous hyphal knots giving the colony a coarse appearance with aging. 
Mycelium colorless, tubular, filamentous, 5-10 um wide, forming hyphal 
segments in older regions. Apical cells 80-450 um long, often unbranched 
before cell division. Conidiophores colorless, unbranched and producing a 
single conidium, arising as upward branches from hyphal knots formed by 
irregular mycelium interweaving, 32.5-110 x 10-15 um. Primary conidia 
colorless, globose to pyriform 17.5-25 um wide, 22.5-32.5um long including 
a basal papilla 7.5-10 um high and 2.5-7.5 um wide. Primary conidia forcibly 
discharged, on water agar forming globose secondary conidia resembling the 
primary spore, 17.5-22.5 x 15-20 um. Zygospores formed between adjacent 
conjugating cells of a hyphal body. Mature zygospores smooth, globose or 
subglobose, 25-31 tm in diameter with wall 1-2 um thick. 


Discussion 

In comparing the morphological characteristics of primary conidiophores 
from cushion mycelium with the known Conidiobolus species, C. sinensis 
resembles C. lichenicola M.C. Sriniv. & Thirum. and C. stromoideus M.C. Sriniv. 
& Thirum. (Srinivasan & Thirumalachar 1962, 1968). Colonies of C. lichenicola 
are distinguished by a pale brownish mycelium with sinuous, lobate hyphae. 
Conidiobolus stromoideus differs from the new species producing edge mycelia 
that are usually branched (rarely branched in C. sinensis) and much shorter 
(12-40um) conidiophores (PLATE 3). 

The phylogenetic tree places the C. sinensis—C. stromoideus clade distant 
from the C. lichenicola clade (PLATE 1), thus reinforcing the morphological 
difference between C. sinensis and C. lichenicola. If C. chlamydosporus is accepted 
as synonymous with C. firmipilleus in accordance with King (1977), there is a 
clear-cut line between intraspecific and interspecific sequence similarity levels: 
98-100% within species and 64-97% among species (TABLE 2). Although 
C. sinensis groups with C. stromoideus, DNA similarity levels between the two 
species fall within the interspecific range (94% (28S rDNA) and 96% (EF-1a)). 
Thus, the phylogenetic analysis supports the morphological identification of 
C. sinensis as a new species differing from C. stromoideus and C. lichenicola. 


Conidiobolus sinensis sp. nov. (China) ... 433 


PLATE 2. Conidiobolus sinensis. A. Colony on PDA after 3 days at 21°C. B. Rarely branched mycelia 
at the margin of colony. C. Primary conidia. D. Primary conidiophores produced from hyphal 
knots. E. Primary conidiophores. FE. Secondary conidia produced singly from the primary conidia. 
G. Mature zygospores. Bars: A = 10 mm, B = 100 um, C-H = 20 um. 


PiaTE 3. A. Conidiobolus sinensis: rarely branched mycelia at the colony edge. B. C. stromoideus: 
moderately branched mycelia at the colony edge. C. C. sinensis: long conidiophores. D. C. stromoideus: 
short conidiophores. Bars: A-B = 100 um, C-D = 20 um. 


434 ... Nie & al. 


Acknowledgments 

We are grateful to Dr. Kathryn E. Bushley (Oregon State University, USA) for 
improving manuscript writing and Dr. Paul Kirk (CABI UK) and Dr. Yu-Cheng Dai 
(Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang, China) for 
reviewing this manuscript. This project was supported by the National Natural Science 
Foundation of China (No. 30770008, No. 31070009, No.31070019) and the Key Science 
Research Project of Anhui Province (No. TD200708). 


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King DS. 1976b. Systematics of Conidiobolus (Entomophthorales) using numerical 
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King DS. 1977. Systematics of Conidiobolus (Entomophthorales) using numerical taxonomy 
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Liu M, Rombach MC, Humber RA, Hodge KT. 2005. What's in a name? Aschersonia insperata: a 
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ISSN (print) 0093-4666 © 2012. Mycotaxon, Ltd. ISSN (online) 2154-8889 


MY COTAXON 


http://dx.doi.org/10.5248/120.437 
Volume 120, pp. 437-441 April-June 2012 


First record of the sequestrate fungus Neosecotium macrosporum 
(Agaricales, Lepiotaceae) from Mexico 


Marcos LIZARRAGA', MARTIN ESQUEDA’ , 
MARIO VARGAS-LUNA’ & GABRIEL MORENO? 


"Dpto. de Ciencias Quimico-Bioldgicas, Instituto de Ciencias Biomédicas, 
Universidad Auténoma de Ciudad Juarez, Anillo Envolvente Pronaf y Estocolmo s/n, 
Cd. Juarez, Chihuahua 32300, México 
*Centro de Investigacién en Alimentacion y Desarrollo, 
A.C. Apartado Postal 1735, Hermosillo, Sonora 83000, México 
°Dpto. de Biologia Vegetal, Facultad de Biologia, Universidad de Alcala, 
Alcala de Henares, Madrid 28871, Spain 


* CORRESPONDENCE TO: esqueda@ciad.mx 


ABSTRACT — Neosecotium macrosporum, previously known only from two USA records, is 
reported for the first time in Mexico. Observations of macro- and microscopic characters 
including for the holotype and the Chihuahua collection are presented. SEM photomicrographs 
illustrating spore ornamentation are included. 


Key worps — Basidiomycota, taxonomy, hypogeous fungi, secotiaceous fungi 


Introduction 

Sequestrate fungi are considered a polyphyletic group with no evolutionary 
relationships whose morphological similarity comes from the adaptation 
to extreme habitats and environmental conditions (Albee-Scott 2007). The 
approximately 1200 species are distributed among eleven orders representing 
the phyla Zygomycota, Ascomycota, and Basidiomycota. Distinguishing 
characteristics are a fleshy, cartilaginous or elastic fruiting body with a persistent 
peridium that encloses the spore producing tissue and thus prevents the easy 
release of the spores (Kendrick 1992). Most sequestrates are mycorrhizogenic 
and serve as a source of food for a number of mammals (Trappe & Claridge 
2003). 

There are few studies of this group in Mexico. The first sequestrate fungus 
recorded for Mexico was collected by Lumholtz (1902) and later designated 
as the holotype of Melanogaster umbriniglebus Trappe & Guzman. Trappe & 


438 ... Lizarraga & al. 


Guzman (1971) described three other new hypogeous species and 13 new 
records for Mexico, Cazares et al. (1992) cited 17 new records, and Cazares et 
al. (2008) described M. minisporus Cazares et al. as a new species and reported 
the first Mexican records of three species of Hysterangium Vittad. 

Neosecotium Singer & A.H. Sm. is a genus characterized by a dry firm 
whitish stipe-columella that extends below a powdery gleba containing few 
fibulae and strongly verrucose spores (Miller & Miller 1988). In comparison, 
Secotium Kunze is characterized by the presence of a volva, a less powdery 
gleba with abundant fibulae, and smooth ellipsoid large spores. Neosecotium is 
represented by two species: N. africanum and N. macrosporum. 


Materials & methods 

The collections were studied according to the standard mycological techniques 
(Cifuentes et al. 1986, Moreno & Manjén 2010). The basidia, spores, peridial hyphae, 
gleba, and columella of specimens mounted in Hoyer’s medium were measured using 
a Nikon Eclipse E200 contrast phase microscope, with some samples examined using a 
Zeiss DSM 950 scanning electron microscope after processing in a Polaron E-2000 for 
1 min at 1.2 Kv and 20 mA, in an argon atmosphere to obtain a 500 A thick deposition 
of gold. The specimens are deposited in the macromycetes collection of the Centro de 
Estudios Superiores de Estado de Sonora (CESUES) and the Universidad Auténoma 
de Ciudad Juarez (UACJ). The holotype was examined using the New York Botanical 
Garden's Virtual Herbarium (NY). Herbarium acronyms follow Thiers (2011). 


Species studied 


Neosecotium macrosporum (Lloyd) Singer & A.H. Sm., 

Madrofio 15(5): 154 (1960). Fics 1-6 

= Secotium macrosporum Lloyd Mycol. Writ. 1: 139. (1903). 
Basip1oMatTa subglobose to pyriform, measuring 11-15 x 7-10 mm. PERIDIUM 
thick, 0.5-0.8 mm, membranaceous, yellowish brown when mature. GLEBA 
composed of tightly packed lamellae, compact or forming pseudo-chambers, 
labyrinthiform in young specimens and powdery in old specimens; yellowish 
and dark brown when young and mature respectively. STIPE-COLUMELLA 
percurrent, whitish, measuring 7-15 x 3-9 mm. PERIDIUM of septate hyphae, 
branched, 2.5-4 um in diam. CoLUMELLA with hyphae similar to the peridium, 
septate, branched, 5-7.5 um in diam. Basrp1a clavate, (25—)28-32(-35) x 3-5 
uum at the base and 9-12 um in the middle and upper part, yellowish brown in 
water; evanescent, leaving amorphous remains when mature. BASIDIOSPORES 
globose, subglobose to ovoid, pseudoamyloid, 10-13(-14) x 8-11 um including 
ornamentation, which is strongly spiny with flat distal end; episporium 2-3 um 
thick, covered by a membrane, which covers the depressions between the spines, 
giving a cracked appearance to the wall as observed under the SEM. PEDICEL 
straight to slightly curved, 2-3 x 2-4 um, narrowing toward the apex. 


Neosecotium macrosporum in Mexico ... 439 


Fics 1-6: Neosecotium macrosporum (CESUES 5600). 
1. Basidiome. 2. Peridium hyphae (LM). 3. Basidium (LM). 4. Spores (LM). 5-6. Spore (SEM). 


SPECIMENS EXAMINED: MEXICO, CurHuanua: Municipality of Juarez, 31°13'53.1"N 
106°30'36.8" W, 1120 masl, in sandy soil near Prosopis glandulosa Torr., 24.V1I.2009, M. 
Lizarraga, C. Salazar, D. Lopez-Pefia, D. Saenz, A.H. Gutiérrez, E. Navarrete (CESUES 
5550); 31°17'54.35"N 106°31'55.45"W, 1303 masl, 17.[X.2010, M. Lizarraga, M. Vargas, 
C. Salazar, D. Saenz (UAC] 1560, CESUES 5600); 19.III.2011, M. Vargas, M. Lizarraga 
(UAC) 1683); 26.11.2011 (UACJ 1684). 


OBSERVATIONS— The Mexican materials were compared with the type and other 
authentic collections of Neosecotium macrosporum in the Virtual Herbarium 
of the New York Botanical Garden (VHNYBG 2011), which allowed us to 


440 ... Lizarraga & al. 


confirm this determination. Although the holotype (NY 809166, from Texas) 
is in a state of deterioration, there are two collections of authentic material 
(NY 809164 and 809165, from North Dakota) identified as N. macrosporum by 
S.M. Zeller. 

Basidiomes of the type and authentic material, in immature stages with 
chambers in an obscure lamellar orientation, wood brown to cinnamon color; 
stipe-columella percurrent, whitish, little projection of the stipe (2-3 mm); spores 
globose, subglobose to ovate, 13-18 or 14-18 x 12-15 um, pseudoamyloid, with 
sterigmal appendage, exosporium smooth becoming cracked into an areolate 
pattern and the fissures produce a warty to echinulate effect; basidia clavate to 
subelliptic-pedicellate, 25-37 x 14-17 um (Lloyd 1903; Singer & Smith 1960). 
The Mexican collections differ in the smaller spore size compared with those 
previously reported. This high variability appears to be inherent to the species 
and has also been reported for taxa close to N. macrosporum. 

According to descriptions by Lloyd (1903) and Singer & Smith (1960), this 
species is easily recognized by its stipe-columella, which extends from the 
base of the gleba to the apex of the peridium, lamelliform gleba and strongly 
verrucose pigmented spores. Although unopened fruiting bodies of Geastrum 
spp. could be confused macroscopically with Neosecotium, the Geastrum is 
easily distinguished by violet spores, a two-layered peridium, and capillitium. 
Based on similar habitat and morphology, Neosecotium might also be confused 
with Endoptychum Czern., which can be distinguished by its smooth spores 
and somewhat larger fruiting bodies (Arora 1986). 

Singer & Smith (1960) regarded Neosecotium as intermediate between the 
Agaricaceae and Lycoperdaceae, based on the variation in color and spore 
ornamentation in some genera in both families. Castellano et al. (2004) 
placed Neosecotium into Lepiotaceae based on recent molecular-supported 
classifications. 

Neosecotium macrosporum has been recorded from two localities in the 
United States of America (Lloyd 1903, Singer & Smith 1960); the present study 
represents a new record for Mexico, and the third for the world. 


Acknowledgments 

The authors are grateful to CONABIO (project GT016) for funding this study. Our 
gratitude to Dr. H. Kreisel and Dr. R. Valenzuela for reviewing the manuscript and their 
useful comments. MVL thanks CONACYT (Mexico) for the award of a fellowship to 
carry out his M.Sc. studies and Aldo Gutierrez (CIAD) for preparing the plates and 
formatting the text. Bianca Delfosse translated the text from the original in Spanish. 


Literature cited 


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Neosecotium macrosporum in Mexico ... 441 


Arora D. 1986. Mushrooms demystified: a comprehensive guide to the fleshy mushrooms. Ten 
Speed Press, Berkeley. 959 p. 

Castellano MA, Trappe JM, Luoma DL. 2004. Sequestrate fungi. 197-213, in: GM Mueller et al. 
(eds). Biodiversity of fungi: inventory and monitoring methods. Elsevier Academic Press, San 
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Cazares E, Garcia J, Castillo J, Trappe JM. 1992. Hypogeous fungi from northern Mexico. Mycologia 
84: 341-359. http://dx.doi.org/10.2307/3760186 

Cazares E, Guevara G, Garcia J, Trappe JM. 2008. Melanogaster minisporus sp. nov., a new 
sequestrate member of the Boletales from Mexico. Revista Mexicana de Micologia 28: 67-69. 

Cifuentes J, Villegas M, Pérez-Ramirez L, Sierra S. 1986. Hongos. 55-64, in: A Lot, F Chiang (eds). 
Manual de herbario. Consejo Nacional de la Flora de México, A.C., México. 

Guevara G, Castellano MA, Garcia J, Cazares E, Trappe JM. 2008. Hysterangium (Hysterangiales, 
Hysterangiaceae) from northern Mexico. Revista Mexicana de Micologia 28: 95-100. 

Kendrick B. 1992. The fifth kingdom. Focus Publishing, Newburyport. 406 p. 

Lumholtz C. 1902. Unknown Mexico. Vol. 1. Charles Scribner’s Sons, New York. 530 p. 

Miller OK Jr, Miller HH. 1988. Gasteromycetes. Morphological and developmental features with 
keys to the orders, families, and genera. Mad River Press, Eureka. 157 p. 

Moreno G, Manjén JL. 2010. Guia de hongos de la Peninsula Ibérica. Ediciones Omega, Madrid. 
1417 p. 

Singer R, Smith AH. 1960. Studies on secotiaceous fungi VII. Secotium and Neosecotium. Madrofio 
15: 152-158. 

Thiers B. 2011. (continuously updated). Index Herbariorum. Part I: the herbaria of the world. New 
York Botanical Garden's Virtual Herbarium. http://sweetgum.nybg.org/ih/ 

Trappe JM, Claridge AW. 2003. Australasian sequestrate (truffle-like) fungi. 15. New species from 
tree line in the Australian Alps. Australasian Mycologist 22: 27-38. 

Trappe JM, Guzman G. 1971. Notes on some hypogeous fungi from Mexico. Mycologia 63: 
317-332. http://dx.doi.org/10.2307/3757764 

VHNYBG. 2011. The C.V. Starr Virtual Herbarium. New York Botanical Garden, International 
Plant Science Center. http://sciweb.nybg.org/science2/vii2.asp [accessed September 2011]. 


ISSN (print) 0093-4666 © 2012. Mycotaxon, Ltd. ISSN (online) 2154-8889 


MYCOTAXON 


http://dx.doi.org/10.5248/120.443 
Volume 120, pp. 443-450 April-June 2012 


New combinations in Lactifluus. 3. 
L. subgenera Lactifluus and Piperati 


A. VERBEKEN’, K. VAN DE PUTTE' & E. DE CRopP’ 


'Ghent University, Department of Biology, Research Group Mycology, 
K.L. Ledeganckstraat 35, B-9000 Gent, Belgium 


CORRESPONDENCE TO: mieke. verbeken@ugent.be 


ABSTRACT — In this last of a series of three papers, new combinations in the genus Lactifluus 
are proposed. This paper treats Lactifluus subg. Lactifluus (an autonymous subgenus) and 
Lactifluus subg. Piperati (proposed as a new subgenus). In Lactifluus subg. Lactifluus, 
six sections are recognized (five of them as new combinations) and 46 new combinations 
are proposed at species level. In Lactifluus subg. Piperati, two sections are recognized (as 
new combinations) and nine new species combinations are proposed. In addition, new 
combinations are proposed for an unassigned section and its single species as well as for 
three unassigned species. 


Key worps — milkcaps, nomenclature, Lactarius, Russulaceae 


Introduction 

This is the third treatment of species formerly treated in Lactarius Pers. 
and now classified in Lactifluus (Pers.) Roussel. Lactifluus subgenera Edules, 
Lactariopsis, and Russulopsis were discussed in the first article (Verbeken et al. 
2012) and Lactifluus subg. Gerardii in the second (Stubbe et al. 2012). 


Taxonomy 


Lactifluus subg. Lactifluus 

This large and diverse subgenus comprises 6 sections. Lactifluus sect. 
Rubroviolascentini, with 2 species, is endemic to tropical Africa. Two sections, 
L. sect. Polysphaerophori and L. sect. Pseudogymnocarpi (each with 8 species) 
are almost completely African, except for one South American species in 
L. sect. Polysphaerophori and one Chinese species in L. sect. Pseudogymnocarpi. 
L. sect. Phlebonemi (11 species) and L. sect. Tomentosi (9 species) also have 
their major distribution in Africa, but contain some species from North and 
Central America, Australia, Europe and Asia. Lactifluus sect. Lactifluus with 


444 ... Verbeken, Van de Putte & de Crop 


Asian, American, and European species is the only section not represented in 
tropical Africa. 

Five species in Lactifluus subg. Lactifluus have already been recombined: 
L. corrugis, L. oedematopus, L. princeps and L. volemus in L. sect. Lactifluus; and 
L. hygrophoroides in L. sect. Tomentosi. 


Lactifluus (Pers.) Roussel, Fl. Calvados, Ed. 2: 66. 1806, subg. Lactifluus 
Type: Agaricus lactifluus L. [= L. volemus (Fr. : Fr.) Kuntze] (see Buyck et al. 2010). 
= Lactarius subg. Lactifluus (Burl.) Hesler & A.H. Sm., N. Am. Species Lactarius: 158. 1979. 
TYPE: Agaricus volemus Fr. : Fr. 


Lactifluus (Pers.) Roussel, Fl. Calvados, Ed. 2: 66. 1806, sect. Lactifluus 
Type: Agaricus lactifluus L. 
= Lactarius subsect. Lactifluini (Burl.) Singer, Ann. Mycol. 40: 114. 1942. 
= Lactarius subsect. Volemi Pacioni & Lalli, Mycotaxon 44: 190. 1992, nom. superfl. 
TYPE: Agaricus volemus Fr. : Fr. 


Lactifluus acicularis (Van de Putte & Verbeken) Van de Putte, comb. nov. 
MycoBank MB 564580 
= Lactarius acicularis Van de Putte & Verbeken, Fungal Diversity 45: 108. 2010. 


Lactifluus austrovolemus (Hongo) Verbeken, comb. nov. 
MycoBaAnk MB 564581 
= Lactarius austrovolemus Hongo, Rep. Tottori Mycol. Inst. 10: 362. 1973. 


Lactifluus corrugis (Peck) Kuntze, Revis. Gen. Pl. 2: 856. 1891. 
= Lactarius corrugis Peck, Annual Rep. New York State Mus. 32: 31. 1880 (“1878”). 


Lactifluus crocatus (Van de Putte & Verbeken) Van de Putte, comb. nov. 
MycoBank MB 564582 
= Lactarius crocatus Van de Putte & Verbeken, Fungal Diversity 45: 112. 2010.. 


Lactifluus distantifolius (Van de Putte, Stubbe & Verbeken) Van de Putte, 
comb. nov. 
MycoBank MB 564583 
= Lactarius distantifolius Van de Putte, Stubbe & Verbeken, 
Fungal Diversity 45: 115. 2010. 


Lactifluus lamprocystidiatus (Verbeken & E. Horak) Verbeken, comb. nov. 
MyYcoBANK MB 564584 
= Lactarius lamprocystidiatus Verbeken & E. Horak, Austr. Syst. Bot. 13: 674. 2000. 
Lactifluus longipilus (Van de Putte, H.T. Le & Verbeken) Van de Putte, comb. nov. 
MycoBank MB 564585 
= Lactarius longipilus Van de Putte, H.T. Le & Verbeken, Fungal Diversity 45: 117. 2010. 


Lactifluus oedematopus (Scop.) Kuntze, Revis. Gen. Pl. 2: 857. 1891. 
= Agaricus oedematopus Scop., Fl. Carniol., Ed. 2, 2: 453. 1772. 
Lactifluus pallidilamellatus (Montoya & Bandala) Van de Putte, comb. nov. 
MycoBank MB 564586 
= Lactarius pallidilamellatus Montoya & Bandala, Cryptog. Mycol. 25: 16. 2004. 


Lactifluus subg. Lactifluus & Piperati ... 


Lactifluus pinguis (Van de Putte & Verbeken) Van de Putte, comb. nov. 
MycosBank MB 564587 
= Lactarius pinguis Van de Putte & Verbeken, Fungal Diversity 45: 119. 2010. 


Lactifluus princeps (Berk.) Kuntze, Revis. Gen. Pl. 2: 857. 1891. 
= Lactarius princeps Berk., Hooker's J. Bot. Kew Gard. Misc. 4: 135. 1852. 


Lactifluus vitellinus (Van de Putte & Verbeken) Van de Putte, comb. nov. 
MycoBank MB 564588 
= Lactarius vitellinus Van de Putte & Verbeken, Fungal Diversity 45: 121. 2010. 


Lactifluus volemus (Fr.: Fr.) Kuntze, Revis. Gen. Pl. 2: 857. 1891. 
= Agaricus volemus Fr.: Fr., Syst. Mycol. 1: 69. 1821. 


Lactifluus sect. Polysphaerophori (Singer) Verbeken, comb. nov. 
MycoBank MB 564589 


= Lactarius sect. Polysphaerophori Singer, Beih. Sydowia 7: 106. 1973. 
Type: Lactarius veraecrucis Singer. 
= Lactarius sect. Gymnocarpi R. Heim ex Verbeken, Mycotaxon 66: 374. 1998. 
Type: Lactarius gymnocarpus R. Heim ex Singer 


Lactifluus albocinctus (Verbeken) Verbeken, comb. nov. 
MycoBank MB 564590 
= Lactarius albocinctus Verbeken, Syst. Geogr. Pl. 70: 182. 2000. 


Lactifluus brunnescens (Verbeken) Verbeken, comb. nov. 
MycoBank MB 564591 
= Lactarius brunnescens Verbeken, Bull. Jard. Bot. Belg. 65: 199. 1996. 


Lactifluus flammans (Verbeken) Verbeken, comb. nov. 
MycoBank MB 564592 
= Lactarius flammans Verbeken, Mycotaxon 55: 539. 1995. 


Lactifluus foetens (Verbeken & Van Rooij) Verbeken, comb. nov. 
MycoBank MB 564593 
= Lactarius foetens Verbeken & Van Rooij, Nova Hedwigia 77: 230. 2003. 


Lactifluus goossensiae (Beeli) Verbeken, comb. nov. 
MycoBank MB 564594 
= Lactarius goossensiae Beeli, Bull. Soc. Roy. Bot. Belgique 60: 165. 1928. 


Lactifluus gymnocarpus (R. Heim ex Singer) Verbeken, comb. nov. 
MycoBank MB 564595 


= Lactarius gymnocarpus R. Heim ex Singer, Pap. Michigan Acad. Sci. 32: 107. 1946. 


Lactifluus tanzanicus (Karhula & Verbeken) Verbeken, comb. nov. 
MycoBank MB 564596 
= Lactarius tanzanicus Karhula & Verbeken, Karstenia 38: 50. 1998. 


Lactifluus veraecrucis (Singer) Verbeken, comb. nov. 
MycosBank MB 564597 
= Lactarius veraecrucis Singer, Beih. Sydowia 7: 104. 1973. 


445 


446 ... Verbeken, Van de Putte & de Crop 


Lactifluus sect. Phlebonemi (R. Heim ex Verbeken) Verbeken, comb. nov. 
MycoBank MB 564598 
= Lactarius sect. Phlebonemi R. Heim ex Verbeken, Mycotaxon 66: 378. 1998. 
Type: Lactarius phlebonemus R. Heim & Gooss.-Font. 
= Lactarius subsect. Luteoli Pacioni & Lalli, Mycotaxon 44: 190. 1992. 
= Lactarius sect. Luteoli (Pacioni & Lalli) Pierotti, Boll. Gruppo Micol. Bres. 48: 54. 2007. 
Tye: Lactarius luteolus Peck 


Lactifluus angustus (R. Heim & Gooss.-Font.) Verbeken, comb. nov. 
MycosBank MB 564599 
= Lactarius angustus R. Heim & Gooss.-Font., Bull. Jard. Bot. Etat 25: 67. 1955. 


Lactifluus arsenei (R. Heim) Verbeken, comb. nov. 
Mycosank MB 564600 
= Lactarius arsenei R. Heim, Candollea 7: 380. 1938, as “arsenii”. 


Lactifluus brunneoviolascens (Bon) Verbeken, comb. nov. 
MycoBank MB 564601 
= Lactarius brunneoviolascens Bon, Doc. Mycol. 1 (2): 45. 1971. 


Lactifluus caribaeus (Pegler) Verbeken, comb. nov. 
MycoBank MB 564602 
= Lactarius caribaeus Pegler, Kew Bull. 33: 617. 1979. 


Lactifluus longivelutinus (X.H. Wang & Verbeken) X.H. Wang, comb. nov. 
MycosBank MB 564603 
= Lactarius longivelutinus X.H. Wang & Verbeken, Nova Hedwigia 83 (1-2): 168, 2006. 


Lactifluus luteolus (Peck) Verbeken, comb. nov. 
MycoBank MB 564604 
= Lactarius luteolus Peck, Bull. Torrey Bot. Club 23: 412. 1896. 


Lactifluus nonpiscis (Verbeken) Verbeken, comb. nov. 
MycoBank MB 564605 
= Lactarius nonpiscis Verbeken, Bull. Jard. Bot. Belg. 65: 204. 1996. 


Lactifluus phlebonemus (R. Heim & Gooss.-Font.) Verbeken, comb. nov. 
MycoBank MB 564606 
= Lactarius phlebonemus R. Heim & Gooss.-Font., Bull. Jard. Bot. Etat 25: 38. 1955. 


Lactifluus pisciodorus (R. Heim) Verbeken, comb. nov. 
MycoBank MB 564607 
= Lactarius pisciodorus R. Heim, Candollea 7: 380. 1938. 


Lactifluus putidus (Pegler) Verbeken, comb. nov. 
MycoBank MB 564608 
= Lactarius putidus Pegler, Kew Bull. 33: 620. 1979. 


Lactifluus rubrobrunnescens (Verbeken, E. Horak & Desjardin) Verbeken, comb. 
nov. 
MycoBank MB 564609 
= Lactarius rubrobrunnescens Verbeken, E. Horak & Desjardin, Sydowia 53: 274. 2001. 


Lactifluus subg. Lactifluus & Piperati ... 


Lactifluus sect. Pseudogymnocarpi (Verbeken) Verbeken, comb. nov. 
MycoBank MB 564610 


= Lactarius sect. Pseudogymnocarpi Verbeken, Mycotaxon 66: 376. 1998. 
Type: Lactarius gymnocarpoides Verbeken 


Lactifluus carmineus (Verbeken & Walleyn) Verbeken, comb. nov. 
MycoBank MB 564611 


= Lactarius carmineus Verbeken & Walleyn, Syst. Geogr. Pl. 70: 190. 2000. 


Lactifluus gymnocarpoides (Verbeken) Verbeken, comb. nov. 
MycoBank MB 564612 


= Lactarius gymnocarpoides Verbeken, Mycotaxon 55: 530. 1995. 


Lactifluus longisporus (Verbeken) Verbeken, comb. nov. 
MycoBank MB 564613 


= Lactarius longisporus Verbeken, Mycotaxon 55: 527. 1995. 


Lactifluus luteopus (Verbeken) Verbeken, comb. nov. 
MycoBank MB 564614 


= Lactarius luteopus Verbeken, Mycotaxon 55: 536. 1995. 


Lactifluus medusae (Verbeken) Verbeken, comb. nov. 
MycoBank MB 564615 


= Lactarius medusae Verbeken, Mycotaxon 55: 532. 1995. 


Lactifluus pseudogymnocarpus (Verbeken) Verbeken, comb. nov. 
MycoBank MB 564616 


= Lactarius pseudogymnocarpus Verbeken, Mycotaxon 55: 523. 1995. 


Lactifluus pumilus (Verbeken) Verbeken, comb. nov. 
MycoBank MB 564617 


= Lactarius pumilus Verbeken, Bull. Jard. Bot. Belg. 65: 205. 1996. 


Lactifluus tenuicystidiatus (X.H. Wang & Verbeken) X.H. Wang, comb. nov. 
MycoBank MB 564618 


= Lactarius tenuicystidiatus X.H. Wang & Verbeken, Nova Hedwigia 83: 173, 2006. 


Lactifluus sect. Rubroviolascentini (Singer) Verbeken, comb. nov. 
MycoBank MB 564619 
= Lactarius subsect. Rubroviolascentini Singer, Ann. Mycol. 40: 114. 1942. 
= Lactarius sect. Rubroviolascentini (Singer) Verbeken, 
Mycotaxon 66: 380. 1998, as “Rubroviolascentes”. 
Type: Lactarius rubroviolascens R. Heim 


Lactifluus denigricans (Verbeken & Karhula) Verbeken, comb. nov. 
MycoBank MB 564620 


= Lactarius denigricans Verbeken & Karhula, Persoonia 16: 219. 1996. 


Lactifluus rubroviolascens (R. Heim) Verbeken, comb. nov. 
MycoBank MB 564621 


= Lactarius rubroviolascens R. Heim, Candollea 7: 377. 1938. 


447 


448 ... Verbeken, Van de Putte & de Crop 


Lactifluus sect. Tomentosi (McNabb) Verbeken, comb. nov. 
MycoBank MB 564622 
= Lactarius sect. Tomentosi McNabb, New Zealand J. Bot. 9: 59. 1971. 
= Lactarius subsect. Clarkeina McNabb, New Zealand J. Bot. 9: 59. 1971. 
Type: Lactarius clarkeae Cleland 
= Lactarius subsect. Rugati Pacioni & Lalli, Mycotaxon 44: 190. 1992, nom. superfl. 
= Lactarius sect. Rugati Verbeken, Mycotaxon 66: 372. 998, 1998, nom. superfl. 
Tye: Lactarius rugatus Kiihner & Romagn. 


Lactifluus clarkeae (Cleland) Verbeken, comb. nov. 
MycoBank MB 564623 


= Lactarius clarkeae Cleland, Trans. & Proc. Roy. Soc. 
S. Australia 51: 302. 1927, as “clarkei”. 


Lactifluus hygrophoroides (Berk. & M.A. Curtis) Kuntze, Revis. Gen. Pl. 2: 857. 
1891. 
= Lactarius hygrophoroides Berk. & M.A. Curtis, Ann. Mag. Nat. Hist., Ser. 3, 4: 293. 1859. 


Lactifluus kivuensis (Verbeken) Verbeken, comb. nov. 
MycoBank MB 564624 
= Lactarius kivuensis Verbeken, Bull. Jard. Bot. Belg. 65: 202. 1996. 


Lactifluus pseudoluteopus (X.H. Wang & Verbeken) X.H. Wang, comb. nov. 
MycoBaAnk MB 564625 
= Lactarius pseudoluteopus X.H. Wang & Verbeken, Nova Hedwigia 83: 171. 2006. 


Lactifluus pseudovolemus (R. Heim) Verbeken, comb. nov. 
MycoBank MB 564626 
= Lactarius pseudovolemus R. Heim, Candollea 7: 378. 1938. 
Lactifluus rubiginosus (Verbeken) Verbeken, comb. nov. 
MycoBank MB 564627 
= Lactarius rubiginosus Verbeken, Bull. Jard. Bot. Belg. 65: 207. 1996. 
Lactifluus rugatus (Kihner & Romagn.) Verbeken, comb. nov. 
MycosBank MB 564628 
= Lactarius rugatus Kihner & Romagn., Bull. Soc. Mycol. France 69: 362. 1954 (“1953”). 
Lactifluus xerampelinus (Karhula & Verbeken) Verbeken, comb. nov. 
MycoBank MB 564629 
= Lactarius xerampelinus Karhula & Verbeken, Karstenia 38: 59. 1998. 
Lactifluus volemoides (Karhula) Verbeken, comb. nov. 
MycoBank MB 564630 
= Lactarius volemoides Karhula, Karstenia 38: 53. 1998. 


Lactifluus subg. Piperati 

This group consists of two sections, one with 9 species described from 
Europe and Asia (but also with records from North America), and the other 
with one American species. The combination Lactifluus piperatus has already 
been proposed. 


Lactifluus subg. Lactifluus & Piperati ... 449 


Lactifluus subg. Piperati Verbeken, subg. nov. 
MycoBank MB 564631 
Pileus pallidus, saepe albus vel albidus, siccus. Pileipellis hyphoepithelium, tenue stratum 
hyphis hyalinis super cellullis globosis. Dermatocystidia interdum presentia. 
TyPE: Agaricus piperatus L.: Fr. 


Lactifluus sect. Piperati (Fr.) Verbeken, comb. nov. 
MycoBank MB 564632 
= Agaricus sect. Piperati Fr., Syst. Mycol. 1: 73. 1821. 
= Lactarius sect. Piperati (Fr.: Fr.) Fr., Epicr. Syst. Mycol.: 338. 1838. 
TyPE: Agaricus piperatus L.: Fr. 


Lactifluus dwaliensis (K. Das, J.R. Sharma & Verbeken) K. Das, comb. nov. 
MycoBank MB 564633 
= Lactarius dwaliensis K. Das, J.R. Sharma & Verbeken, Mycotaxon 88: 334. 2003. 


Lactifluus glaucescens (Crossl.) Verbeken, comb. nov. 
MyYcoBANK MB 564634 
= Lactarius glaucescens Crossl., Naturalist, J. Nat. Hist. N. England 1900(516): 5. 1900. 
= Lactarius piperatus var. glaucescens (Crossl.) Hesler & A.H. 
Sm., N. Amer. Species Lactarius: 186. 1979 


Lactifluus leuacophaeus (Verbeken & E. Horak) Verbeken, comb. nov. 
MycoBank MB 564635 
= Lactarius leucophaeus Verbeken & E. Horak, Austr. Syst. Bot. 12: 768. 1999. 


Lactifluus novoguineensis (Henn.) Verbeken, comb. nov. 
MycoBank MB 564636 
= Lactarius novoguineensis Henn., Bot. Jahrb. Syst. 25: 503. 1898. 


Lactifluus olivescens (Verbeken & E. Horak) Verbeken, comb. nov. 
MycoBank MB 564637 
= Lactarius olivescens Verbeken & E. Horak, Austr. Syst. Bot. 13: 678. 2000. 


Lactifluus paleus (Verbeken & E. Horak) Verbeken, comb. nov. 
MycoBank MB 564638 
= Lactarius paleus Verbeken & E. Horak, Austr. Syst. Bot. 12: 771. 1999. 


Lactifluus piperatus (L. : Fr.) Kuntze, Revis. Gen. Pl. 2: 857. 1891. 
= Agaricus piperatus L.: Fr., Sp. Pl.: 1173. 1753. 
= Lactarius piperatus (L. : Fr.) Pers., Tent. Disp. Meth. Fung.: 64. 1797. 
= Galorrheus piperatus (L.: Fr.) Fr., Stirp. Agri Femsion. 3: 57. 1825. 


Lactifluus roseophyllus (R. Heim) De Crop, comb. nov. 
MycoBank MB 564639 
= Lactarius roseophyllus R. Heim, Rev. Mycol. (Paris) 30: 237. 1966 (“1965”). 


Lactifluus subpiperatus (Hongo) Verbeken, comb. nov. 
MyYcoBANK MB 564647 
= Lactarius subpiperatus Hongo, Mem. Fac. Liberal Arts 
Shiga Univ., Nat. Sci. 15: 46. 1964. 


450 ... Verbeken, Van de Putte & de Crop 


Lactifluus sect. Allardii (Hesler & A.H. Sm.) De Crop, comb. nov. 
MycosBank MB 564640 


= Lactarius sect. Allardii Hesler & A.H. Sm., N. Amer. Species Lactarius: 207. 1979. 
Type: Lactarius allardii Coker 


Lactifluus allardii (Coker) De Crop, comb. nov. 
MycoBank MB 564641 


= Lactarius allardii Coker, J. Elisha Mitchell Sci. Soc. 34: 12. 1918. 


Unassigned taxa 
The following section has not yet been assigned to a subgenus. Only one 
species, described from tropical Africa, is known. 


Lactifluus sect. Aurantiifolii (Verbeken) Verbeken, comb. nov. 
MycoBank MB 564642 


= Lactarius sect. Aurantiifolii Verbeken, Mycotaxon 77: 441. 2001. 
Tye: Lactarius aurantiifolius Verbeken 


Lactifluus aurantiifolius (Verbeken) Verbeken, comb. nov. 
MyYcoBANK MB 564643 
= Lactarius aurantiifolius Verbeken, Bull. Jard. Bot. Belg. 65: 197. 1996. 


The following species have an uncertain systematic position, but morphological 
and/or molecular data support their placement in Lactifluus. 


Lactifluus caperatus (R. Heim & Gooss.-Font.) Verbeken, comb. nov. 
MyYcoBANK MB 564644 


= Lactarius caperatus R. Heim & Gooss.-Font., Bull. Jard. Bot. Etat 25: 36. 1955. 


Lactifluus cocosmus (Van de Putte & De Kesel) Van de Putte, comb. nov. 
MyYcoBANK MB 564645 


= Lactarius cocosmus Van de Putte & De Kesel, Cryptog. Mycol. 30: 40. 2009. 


Lactifluus subclarkeae (Grgur.) Verbeken, comb. nov. 
MycoBank MB 564646 


= Lactarius subclarkeae Grgur., Larger Fungi S. Australia: 63, 1997. 


Acknowledgments 
The authors acknowledge Scott Redhead and Shaun Pennycook for valuable 
comments and for reviewing the manuscript. 


Literature cited 

Buyck B, Hofstetter V, Verbeken A, Walleyn R. 2010. Proposal to conserve Lactarius nom. cons. 
(Basidiomycota) with a conserved type. Taxon 59: 295-296. 

Stubbe D, Wang X-H, Verbeken A. 2012. New combinations in Lactifluus. 2. L. subg. Gerardii. 
Mycotaxon 119: 483-485. http://dx.doi.org/10.5248/119.483 

Verbeken A, Nuytinck J, Buyck B. 2012. New combinations in Lactifluus. 1. L. subgenera Edules, 
Lactariopsis, and Russulopsis. Mycotaxon 118: 455-458. http://dx.doi.org/10.5248/118.447 


ISSN (print) 0093-4666 © 2012. Mycotaxon, Ltd. ISSN (online) 2154-8889 


MY COTAXON 


http://dx.doi.org/10.5248/120.451 
Volume 120, pp. 451-460 April-June 2012 


Exobasidium ferrugineae sp. nov., associated with hypertrophied 
flowers of Lyonia ferruginea in the southeastern USA 


AARON H. KENNEDY’, NISSE A. GOLDBERG? & ANDREW M. MINNIS? 


‘National Identification Services, USDA-APHIS-PPQ-PHP, 
10300 Baltimore Ave., B 580, Beltsville, MD, 20705, USA 
Jacksonville University, Dept. of Biology and Marine Science, 
2800 University Blvd. North, Jacksonville, FL 32211, USA 
°Center for Forest Mycology Research, Northern Research Station, 
USDA-Forest Service, One Gifford Pinochet Drive, Madison, WI 53726, USA 
* CORRESPONDENCE TO: amminnis@fs.fed.us 


Apstract — Exobasidium ferrugineae, associated with hypertrophied flowers and less 
commonly leaves of Lyonia ferruginea (rusty staggerbush), is formally described here as a 
new species. Morphological and DNA sequence (ITS, nLSU) data are provided. Phylogenetic 
analyses confirm that it is not conspecific with any species of Exobasidium represented by 
existing DNA sequence data. A key to North American species of Exobasidium on Lyonia is 
presented. 


Key worps — Basidiomycota, Ericaceae, Exobasidiales, Exobasidiomycetes, plant pathogen 


Introduction 

Exobasidium Woronin (Exobasidiales, Exobasidiomycetes) is a basidio- 
mycetous genus associated with diseases of ericaceous plants commonly 
characterized by formation of galls on leaves, shoots, and flowers (Burt 1915, 
Savile 1959, Nannfeldt 1981). Early authors named species on the basis of 
symptomatology and host association, whereas monographers, including Burt 
(1915) and Savile (1959), advocated broader taxonomic concepts. These authors 
suggested that symptoms were variable, overlapping, and dependent on time and 
environmental conditions. Furthermore, fungal morphology was not definitive 
for species recognition and usually poorly known, and host associations are 
not supported by inoculation and cross-inoculation experiments. According to 
these authorities, only a small number of species were accepted in Exobasidium 
and the citation of numerous synonyms was deemed unreliable (Burt 1915, 
Savile 1959). Nannfeldt (1981) reviewed a large amount of data and literature 


452 ... Kennedy, Goldberg & Minnis 


with special emphasis on life cycles and symptoms. He concluded that species 
of Exobasidium, though often quite similar morphologically, are rigidly host- 
limited taxa. Recent molecular studies including those by Begerow et al. (2002) 
and Piatek et al. (2012) support Nannfeldt’s hypotheses on the importance of 
host association and host specificity. 

In April 2011, an unusual flower of Lyonia ferruginea (Walter) Nutt. (rusty 
staggerbush; Ericaceae) that was infected and highly transformed by a fungus 
was found in Florida. Microscopic examination confirmed that a species 
of Exobasidium was growing on the hypertrophied flower. This species was 
subsequently characterized with morphological and DNA sequence (ITS, 
nLSU) data. Careful comparison with known species using historical literature, 
herbarium specimens, and phylogenetic analyses revealed that it represents an 
undescribed taxon that is described herein. A key to North American species 
of Exobasidium on Lyonia is presented in order to facilitate identification and 
promote further study of Exobasidium from North America. 


Materials & methods 


Morphology 

Observations and measurements of microscopic characters were made from crush 
mounts of material scraped from the hymenial surface that covered the flower. Material 
was rehydrated and viewed in 3% KOH, and occasionally phloxine solution was used 
to improve the visibility of fungal structures (Largent et al. 1977). Sterigmata are not 
included in length measurements of basidia. Voucher specimens were deposited into the 
US. National Fungus Collections (BPI). 


Molecular data collection & phylogenetic analysis 

DNA was extracted from scraped hymenial surface material from the infected flower 
of L. ferruginea (N.A. Goldberg, s.n.; BPI 882571) using Qiagen’s DNeasy Plant Mini Kit 
(Germantown, MD). Ribosomal DNA sequence data were generated from the internal 
transcribed spacer region (ITS; ITS1, 5.88, ITS2) and the nuclear encoded large subunit 
(nLSU) using the primer pairs ITS5/ITS4 (White et al. 1990) and NL1/NL4 (O’Donnell 
1992), respectively. GoTaq (Promega, Madison, WI) and associated standard reagents 
were used for PCR following the manufacturer recommendations including 2.0 mM 
MgCl. Standard thermal cycling was used including an annealing temperature of 55°C 
for the ITS and 50°C for the nLSU. PCR products were fluorescently labeled using 
respective forward and reverse PCR primers and the BigDye v3.1 dye terminator kit 
(Applied Biosystems; Foster City, CA). The purified products were then sequenced on 
an ABI 3730 automated DNA sequencer. Electropherograms were edited in Geneious 
Pro v.5 (Drummond et al. 2010). Resulting consensus sequences were submitted to 
GenBank (http://www.ncbi.nlm.nih.gov). 

BLAST searches were conducted independently with the ITS and nLSU sequences 
against GenBank using the megablast algorithm. Taxon sampling for phylogenetic 
analyses was primarily determined by selecting the top hits from each search, sorted 
by Max Score; the phylogenetic results of Pigtek et al. (2012); and morphological and 


Exobasidium ferrugineae sp. nov. (USA) ... 453 


host association similarity. The ITS and nLSU sequences for each taxonomic unit were 
derived from the same isolate. 

Multiple sequence alignments of the ITS and nLSU were conducted using 
MUSCLE v3.6 (Edgar 2004) within Geneious Pro v5 (Drummond et al. 2010) and 
then concatenated. Although nLSU data were available for all taxa included in 
the concatenated alignment, ITS data were unavailable for Exobasidium karstenii, 
E. oxycocci, E. pieridis, and E. sundstroemii and therefore treated as missing data. The 
best-fit model of DNA sequence evolution was independently determined for ITS 
and nLSU in MrModeltest v2.2 (Nylander 2004) according to the Akaike Information 
Criterion (AIC; Posada & Buckley 2004). The GTR+I+G model was selected for both 
data sets and implemented in the following analysis. The concatenated alignment was 
analyzed phylogenetically with Bayesian Inference (BI) in MrBayes v3.1.2 (Huelsenbeck 
& Ronquist 2001; Ronquist & Huelsenbeck 2003) leaving all other parameters as default. 
The posterior probability (pp) distribution of trees was estimated from two independent 
Markov Chain Monte Carlo (MCMC) simulations of 3 million generations, sampling 
trees every 100 generations until the standard deviation of split frequencies reached 0.01. 
The burn-in was determined using the program Tracer v1.5 (Rambaut & Drummond 
2007). A 50% majority rule consensus tree was constructed from the remaining trees in 
FigTree v1.3.1 (Rambaut 2009). 

The concatenated alignment was also analyzed using Maximum Likelihood (ML). 
This analysis was conducted using RAxML (Stamatakis 2006) via the on-line CIPRES 
(Cyberinfrastructure for Phylogenetic Research) Science Gateway V. 3.1 (http://www. 
phylo.org/sub_sections/portal/; Miller et al. 2010) with the RAxML-HPC BlackBox 
(7.2.8) tool. One thousand bootstrap (bs) replicates were run and all other parameters 
were left as default. 


Results 

Alignment of the ITS resulted in a matrix of 595 nucleotide positions, 296 
of which were variable (49.7%) and 156 of which were parsimony informative 
(26.2%). Alignment of the nLSU resulted in a matrix of 555 nucleotide 
positions, 146 of which were variable (26.3%) and 71 of which were parsimony 
informative (12.8%). 

The phylogenies resulting from the BI and ML analyses were entirely 
congruent with the exception of the E. pieridis/E. ferrugineae clade, which 
occupied a different position in the most optimal ML tree vs. the BI consensus 
tree (Fic. 1). This is an insignificant observation considering the position of 
this clade is entirely unsupported in the ML bootstrap tree and received very 
low posterior probability in the BI tree. Each analysis revealed high support 
(pp = 0.99, bs = 99%) for the E. pieridis/E. ferrugineae clade, revealing a close 
relationship between these species and demonstrating that E. ferrugineae is a 
species distinct from E. vaccinii (Fic. 1). 

The relationship between Exobasidium ferrugineae and E. karstenii was 
of particular interest because E. karstenii was among the top hits for nLSU. 


454 ... Kennedy, Goldberg & Minnis 


Tilletiopsis pallescens AY259059/AY 272004 
Laurobasidium hachijoense AB180359/AB 177562 
E. sundstroemii N.A./AF487396 
E. pieridis-ovalifoliae AB180367/AB 1177601 
0.67/* ¥ p= E. bisporum AB180364/AB177598 
i a AB180368/AB177596 
E. darwinii FJ896133/FJ896134 
eR . pachysporum AB180352/AB177573 
c E. arescens FJ896135*/FJ896136 
E. rostrupii FJ896132*/FJ896137 
E. inconspicuum AB180347/AB177556 
0.96/97 E. cylindrosporum AB245089/AB 176713 
0.62/*}* E. pulchrum EU692776*/EU692795 
0.67/"t_— E, pentasporium AB180334/AB177581 
0.75/L E. nobeyamense AB180331/AB177582 
O.BRIe E. otanianum AB180345/AB177593 
E. miyabei AB180330/AB177579 
E. japonicum AB180315/AB177548* 
0 Yoyo E. shiraianum AB180336/AB177549 
E. rhododendri DQ667153/DQ667151* 
0.9/72L_ E. woronichinii AB180348/AB177557 
1/100 E. camelliae AB176712/AB180317 
1/100 E. gracile DQ663700/DQ663699 
0.99/82 E. reticulatum AB180377/AB 180381 
0.56/** E. symploci-japonicae AB180678/AB 178255 
OY p> E. ferrugineae JQ611710/IQ611711_ <<—— 
E. pieridis N.A./AB177575 
OVE. kishianum AB180353/AB177577 
, E. kishianum AB180354/AB177555 
099° L 1098/68. Fe vaccinii AB180362/AB177560 
0.63/* E. karstenii N.A./AF487389* 
E. oxycocci N.A./AF487391 


0.9 


0.94/84 


0.69/* 


0.07 


Fic. 1. A phylogenetic summary of relationships among Exobasidium species resulting from 
Bayesian Inference (BI) and Maximum Likelihood (ML) analyses of a combined ITS/nLSU data 
matrix. This phylogram is the resulting 50% majority rule consensus tree from the BI analysis 
with corresponding posterior probabilities (pp) and appended bootstrap (bs) values from the 
ML analysis on each branch (pp/bs). One asterisk indicates a bs value < 50%, while two asterisks 
indicates a clade that was not recovered in the most optimal ML tree. The hatch marks on the 
“Laurobasidium hachijoense” nom. prov. in GenBank [= E. hachijoense] branch indicate that it was 
shortened for presentation purposes from 8.5 times the scale bar. GenBank accession numbers 
are provided after each taxon name (ITS/nLSU). Asterisks here indicate top BLAST hits for the 
associated marker. The arrow indicates the position of E. ferrugineae (holotype, BPI 882571). 


However, our phylogenetic results suggested, albeit with very weak support 
(pp=0.53, bs = 68), that E. karstenii is most closely related to E. vaccinii. Again, 
the relationship of this clade to others was unsupported. 


Exobasidium ferrugineae sp. nov. (USA) ... 455 


Taxonomy 


Exobasidium ferrugineae Minnis, A.H. Kenn. & N.A. Goldberg, sp. nov. Fics. 2-4 
MycoBANnk 564773 
Characterized by its association with hypertrophied flowers of Lyonia ferruginea. 
TYPE: USA. Florida: Jacksonville, Jacksonville Arboretum, Rosemary Ridge Trail, on 


flower of Lyonia ferruginea, 18.IV.2011, leg. N.A. Goldberg, s.n., (Holotype BPI 882571; 
GenBank accession nos., ITS JQ611710, nLSU JQ611711). 


Erymo oey: The species epithet is derived from the host, Lyonia ferruginea. 


HYMENIUM consisting of a thin white layer on hypertrophied flowers having 
all parts proportionally enlarged, infected flowers up to approx. 6 cm long, or 
less commonly on leaves that may also be hypertrophied. Basip1a 37-54.5 x 
6.5-8.5 um, cylindrical to cylindrical-clavate, 3-5 sterigmate, thin walled, 
hyaline, often with granular contents. STERIGMATA approx. 4.5-6.5 x 1-2 
um, typically slightly wider towards base. BAsipriospoREs 13.5-19 x 4-6.5 
um, ellipsoid to musiform, with a prominent apiculus, walls thin and smooth, 
initially with one more or less median septum at maturity, later with up to six 
septa, hyaline. GERMINATION via germ tubes at both ends of basidiospores. 
ConipiA 4-9.5 x 1-2.5 um, bacilliform, ellipsoid or slightly clavate, walls thin 
and smooth, aseptate, hyaline, contents often with a few scattered guttules. 

HABITAT & DISTRIBUTION— ‘This species is known from hypertrophied 
flowers and less commonly hypertrophied leaves of Lyonia ferruginea 
(Ericaceae). It is known from Florida and Georgia, and is predicted to occur 
in South Carolina, and throughout the narrow, native range of its host (USDA 
2011). 


ADDITIONAL SPECIMENS EXAMINED— USA. FLoripa: on flower of Lyonia ferruginea, 
leg. Chapman, s.n., Mo. Bot. Gard. Herb. 44409 (BPI 292003 as Exobasidium vaccinii); 
Dunedin, on flower of Lyonia ferruginea, 16.1V.1900, leg. S.M. Tracy, SMT 7159, Mo. 
Bot. Gard. Herb. 4962 (BPI 292004 as E. vaccinii); Jacksonville, Jacksonville Arboretum, 
Rosemary Ridge Trail, on leaves of Lyonia ferruginea, 06.V.2011, leg. N.A. Goldberg, s.n. 
(BPI 882572); additional collections on Lyonia ferruginea made by N.A. Goldberg from 
the area of the type locality, Rosemary Ridge Trail: 18.IV.2011 (BPI 882566, BPI 882567, 
BPI 882568, BPI 882569, BPI 882570, BPI 882580, BPI 882582, BPI 882583), 06.V.2011 
(BPI 882573, BPI 882574, BPI 882575, BPI 882576, BPI 882577, BPI 882578, BPI 882579, 
BPI 882581). Georetra: Brunswick, on flower of Lyonia ferruginea, 01.V1.1901, leg. 
W. Trelease, s.n., Mo. Bot. Gard. Herb. 4955 (BPI 292005 as E. vaccinii). 


CoMMENTS— Burt (1915) studied collections of hypertrophied flowers of Lyonia 
ferruginea caused by Exobasidium ferrugineae and concluded that the species 
was conspecific with E. vaccinii. Burt (1915) also commented on the fact that 
the hypertrophied flowers were much larger than those of L. mariana found 
in association with E. peckii Halst., which he also concluded was a synonym 
of E. vaccinii. However, the results presented here show that E. ferrugineae is 
distinct from E. vaccinii. Exobasidium vaccinii has been shown to be restricted 


456 ... Kennedy, Goldberg & Minnis 


Fr, - i 


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my ‘aig ape 


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Fic. 2. Exobasidium ferrugineae associated with hypertrophied flowers of Lyonia ferruginea. 


Note uninfected flowers in upper image for comparison. All flower parts are proportionally 
enlarged. 


to Vaccinium, likely just Vaccinium vitis-idaea (Nannfeldt 1981, Begerow et al. 
2002, Piatek et al. 2012). 


Exobasidium ferrugineae sp. nov. (USA) ... 457 


Fic. 3. Exobasidium ferrugineae associated with hypertrophied leaves of Lyonia ferruginea. 


Several species of Exobasidium are known from Lyonia (Farr & Rossman 
2011) and eight of these were described originally from this host genus. 
Five Asian species, E. kunmingense Zhen Ying Li & L. Guo (Li & Guo 2009), 
E. lyoniae Zhen Ying Li & L. Guo (Li & Guo 2006), E. ovalifoliae Zhen Ying Li 
& L. Guo (Li & Guo 2008), E. pieridis Henn. (Hennings 1903), and E. pieridis- 
ovalifoliae Sawada (Sawada 1931), are distinguished from E. ferrugineae by 
their occurrence on L. ovalifolia and their association that is confined to leaf 
diseases. There are three previously described species on Lyonia from North 
America. Exobasidium andromedae Peck (Peck 1874) and E. fawcettii Massee 
(Massee 1908) occur on different hosts than E. ferrugineae and are associated 
with leaf diseases. At times, E. andromedae may produce unique bag galls where 
developing leaves are transformed into hollow bags (Peck 1874, Burt 1915). 
Exobasidium peckii is similar to E. ferrugineae as it infects both flowers and 
leaves (Halstead 1893, Stewart 1896), but it is distinguished by its occurrence 
on Lyonia mariana and a lesser amount of hypertrophy on flowers. Based 
on our morphological examination of a collection of E. peckii (USA: New 
York, Hicksville, Long Island, on flower of Lyonia mariana, 25.V1.1919, leg. 
G.P. Clinton, s.n. (BPI 292023 as E. vaccinii)), this species is nearly 
indistinguishable morphologically from E. ferrugineae. However, E. peckii 
differs in the basidiospores with none having been observed with more than 5 
septa along with typically fewer septa per basidiospore and the conidia having 
a tendency towards an allantoid shape. 

Our phylogenetic analyses indicate that E. ferrugineae is distinct from all 
species with existing ITS and nLSU sequence data in GenBank (data were 


458 ... Kennedy, Goldberg & Minnis 


Fic. 4. Exobasidium ferrugineae (holotype, BPI 882571). 
A-B. Basidia. C. Basidiospore. D. Conidia. Scale bars are 20 um for A-B, 10 um for C-D. 


unavailable for species on Lyonia including E. andromedae, E. fawcettii, E. 
kunmingense, E. lyoniae, E. ovalifoliae, E. peckii) including the isolates that 
are labeled E. pieridis and E. pieridis-ovalifoliae from Lyonia neziki. ‘The close 
relationship between E. ferrugineae and E. pieridis is noteworthy because of 
their highly discontinuous geographic ranges. We suggest that this geographic 
isolation has led to reproductive isolation and that the relationship between 
E. ferrugineae and E. pieridis is a product of the tendency for host specificity 
in Exobasidium, in this case with Lyonia, and divergence in host range 
relative to their recent common ancestor. Therefore, their well-supported 
sister-relationship found here suggests recent common ancestry, but not 
conspecificity. Unfortunately, ITS data were unavailable for E. pieridis, 
resulting in missing data for combined analysis. This is significant because the 
ITS region is more informative than nLSU primarily because it more rapidly 
accumulates substitutions among species. This is evidenced by the fact that the 


Exobasidium ferrugineae sp. nov. (USA) ... 459 


ITS region contains twice the number of variable and informative characters 
among Exobasidium species. Therefore, support for this relationship may be 
artificially high in this combined analysis due to missing ITS data for E. pieridis. 
Further sampling will be needed to address the complex patterns of speciation 
including potential co-speciation in Exobasidium as discussed by Begerow et al. 
(2002) and Piatek et al. (2012). 


Key to Exobasidium on Lyonia of North America 


1. Onhligustring, with bag Sallss ea ghee gba 2g heer Ba bee Babar 3 ghee E. andromedae 
}- On. other species of: Lyvonta, bac.ealls absent oats gta! edna ads a ddanaie « enwictas. date 2 
2. On L. jamaicensis, with hypertrophied leaves ...................0000- E. fawcettii 
2. On other species of Lyonia, associated with flowers & occasionally leaves ........ 3 


3. On L. mariana, with hypertrophied flowers relatively small (up to approx. 
2-<longet);some- conidia allantoid tos .% 06 wea a ole bia ew eget w a E. peckii 
3. On L. ferruginea, with hypertrophied flowers relatively large (up to approx. 
5. xlonger);conidia not, allantoid. we. 62 west wate s wees 2 wanes 2 E. ferrugineae 


Acknowledgments 

The authors thank Dr. Lori M. Carris of Washington State University and Dr. Alistair 
R. McTaggart of Louisiana State University for their expertise and time in reviewing 
this work. Dr. Amy Y. Rossman of Systematic Mycology and Microbiology Laboratory 
kindly provided additional helpful suggestions. 


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from http://nt.ars-grin.gov/fungaldatabases/fungushost/fungushost.cfm 

Halsted BD. 1893. Notes upon a new Exobasidium. Bull. Torrey Bot. Club 20: 437-440. 
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Hennings P. 1903. Fungi japonica. III. Bot. Jahrb. Syst. 32: 34-46. 

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Largent D, Johnson D, Watling R. 1977. How to identify mushrooms to genus III: Microscopic 
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Li Z, Guo L. 2006. A new species and a new Chinese record of Exobasidium (Exobasidiales) from 
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MY COTAXON 


http://dx.doi.org/10.5248/120.461 
Volume 120, pp. 461-463 April-June 2012 


Passalora aseptata, a new cercosporoid fungus 
from northeastern Uttar Pradesh, India 


RAGHVENDRA SINGH , BALMUKUND CHAURASIA, 
KALAWATI SHUKLA & PARMATMA PRASAD UPADHYAYA 


Department of Botany, D.D.U. Gorakhpur University, Gorakhpur, U.P. - 273 009, India 
*CORRESPONDENCE TO: drsinghtaxon@gmail.com 


AxsstRact — The anamorphic fungus Passalora aseptata sp. nov., found on Terminalia 
bellirica (Combretaceae), in the Chowk Forest, Mahrajganj, U.P., India, is described and 
illustrated. 


Key worps — biodiversity, foliar diseases, phytopathogenic fungi, taxonomy 


Introduction 

During an investigation of plant diseases, fungi were collected on living 
leaves from forests in northeastern Uttar Pradesh during 2008. Among the 
collections an undescribed cercosporoid species was found. Based on pigmented 
conidia and conidiophores as well as conidiogenous cells with thickened scars, 
this fungus can be assigned to Passalora as circumscribed by Crous & Braun 
(2003). 


Materials & methods 

Specimens with disease symptoms of cercosporoid fungi on living leaves were 
collected during the course of field trips. Detailed observations of morphological 
characters were carried out by means of an Olympus CX31 light microscope using oil 
immersion (1000x). Specimens for microscopic observation were prepared by hand 
sectioning. Water and lactophenol were used as mounting media. Measurements were 
made of 30 conidia, hila, and conidiophores and of 15 stromata. Line drawings were 
prepared at a magnification of 1000x. Morphotaxonomic determinations were made 
with the help of current literature and available resident expertise. The holotype has been 
deposited in Herbarium Cryptogamiae Indiae Orientalis (HCIO), Indian Agricultural 
Research Institute, New Delhi, India; and an isotype was retained in the herbarium of 
Department of Botany, D.D.U. Gorakhpur University (GPU) for further reference. 


462 ... Singh & al. 


Taxonomy 


Passalora aseptata R. Singh, Chaurasia, K. Shukla & Upadhyaya, sp. nov. 
MycoBank MB 519057 FIG. 1 
Differs from all other Passalora species in aseptate conidia and conidiophores. 


Type: India, Uttar Pradesh, Mahrajganj, Chowk Forest, on living leaves of Terminalia 
bellirica (Gaertn.) Roxb. (Combretaceae), Jan. 2008, coll. Raghvendra Singh, (holotype, 
HCIO 48785; isotype, GPU KSR-395). 


ErymMo_oey: Latin, aseptata refers to aseptate conidia and conidiophores. 


Infection spots hypogenous, discrete and initially vein-limited but later 
coalescing to become irregular and more or less necrotic, light brown, 
spreading over entire lower leaf surface. Colonies hypophyllous, effuse, 
velvety, light brown. Mycelium internal. Stromata well developed, erumpent, 
pseudoparenchymatous, substomatal, 26-43 x 22-31 um. Conidiophores 
arising from stromata, in fascicles of 10-12, macronematous, simple, erect to 
procumbent, straight to flexuous, cylindrical, unbranched, smooth, thin-walled, 


Fic. 1. Passalora aseptata (holotype). 
a: symptoms; b: stroma, conidia, and conidiophores. 
(Scale bars: a = 20 mm, b = 20 um). 


Passalora aseptata sp. nov. (India) ... 463 


light brown to olivaceous-brown, aseptate, 29-48 x 4-7 um. Conidiogenous 
cells integrated, terminal, monoblastic, scars conspicuously thickened, 
darkened, 0.7-1.5 um wide. Conidia solitary, dry, acropleurogenous, simple, 
obclavate-cylindrical, erect, straight to curved, sometimes rostrate, aseptate, 
thin-walled, smooth, light brown to olivaceous-brown, 40-70 x 4-9 um, hila 
slightly thickened, 0.7-1.5 um wide. 


REMARKS — No species of Passalora Fr. has previously been described with 
aseptate conidia and conidiophores. A survey of literature also indicates that no 
species of Passalora has previously been described on a host of genus Terminalia 
or family Combretaceae. 

Prathigada terminaliae (Syd.) B. Sutton (Sutton 1994), Pseudocercospora 
brevis B. Sutton (Sutton 1994), P combretacearum var. minima B. Sutton 
(Sutton 1994), P combreti A.K. Singh & Kamal (Singh & Kamal 1987), and 
Scolecostigmina combreti (J. Kranz) U. Braun (Braun 1999) have been reported 
on the same host species. Prathigada terminaliae is different due to the presence 
of polyblastic, cicatrized, conidiogenous cells with thin, flat conidiogenous loci 
and rostrate conidia having smooth or rugulose, multiseptate (septa often rather 
thick and dark) and unequally pigmented cells. All Pseudocercospora species 
differ from Passalora aseptata in having euseptate conidia and conidiogenous 
cells without thickened scars. Scolecostigmina combreti is readily separated by 
its formation of sporodochia, annellations on conidiophores and truncate base 
of conidia. 

Passalora bacilligera (Mont. & Fr.) Mont. & Fr., type species of Passalora, and 
all other species of this genus are easily distinguished from the novel species in 
having septate conidia. 


Acknowledgments 

The authors are grateful to Prof. Dr. Uwe Braun and Dr. Eric H.C. McKenzie for 
reviewing the manuscript. We also express our deep thanks to Dr. Shaun Pennycook 
for nomenclatural review. We are much obliged to Prof. Dr. Kamal for the identification 
of host plant and editing the manuscript. Authors’ thanks are also due to the Curator, 
HCIO, New Delhi, for accepting the holotype specimens and providing an accession 
number thereof. 


Literature cited 

Braun U. 1999. Some additional species of Scolecostigmina. Schlechtendalia 3: 33-42. 

Crous PW, Braun U. 2003. Mycosphaerella and its anamorphs: 1. Names published in Cercospora 
and Passalora. CBS, Utrecht, The Netherlands, Fungal Biodiversity Centre. 571 p. 

Singh AK, Kamal. 1987 [“1986”]. Fungi of Gorakhpur. XXXIX. Pseudocercospora. Kavaka 14: 
Pier ey 

Sutton BC. 1994. IMI Descriptions of Fungi and Bacteria, Nos 1181-1190. Mycopathologia 125(1): 
43-64. http://dx.doi.org/10.1007/BF01103975 


ISSN (print) 0093-4666 © 2012. Mycotaxon, Ltd. ISSN (online) 2154-8889 


MY COTAXON 


http://dx.doi.org/10.5248/120.465 
Volume 120, pp. 465-469 April-June 2012 


First records of Craterium aureonucleatum, 
Perichaena quadrata, and Physarum mutabile in Italy 


RICCARDO COMPAGNO, ALFONSO LA ROSA, & GIUSEPPE VENTURELLA* 


Dipartimento di Biologia Ambientale e Biodiversita, Via Archirafi 38, I-90123 Palermo, Italy 
*CORRESPONDENCE TO: giuseppe.venturella@unipa. it 


ABsTRACT — Three species of Mycetozoa, new for Italy, were identified in garrigues and 
maquis ecosystems on the volcanic island of Pantelleria (Sicily). The finding of Craterium 
aureonucleatum, Perichaena quadrata, and Physarum mutabile widens their distribution in 
Europe and identifies new substrata. 


Key worps — biodiversity, Mediterranean area, ecology 


Introduction 

Within a wide-scoped biodiversity census, field investigations were carried 
out on the island of Pantelleria (Trapani province, southwest Sicily, Italy). In 
his checklist of Mycetozoa in the Mediterranean area, Lado (1994) reported 
421 taxa, of which 164 were recorded from Italy. To increase the knowledge 
of mycetozoan diversity in Italy, we focused primarily on the collection 
of Mycetozoa in Mediterranean forest ecosystems characterizing the plant 
landscape of Pantelleria (Gianguzzi 1999). The Erico arboreae-Quercetum ilicis 
typicum plant association and Myrtus communis facies (Brullo et al. 1977) 
is dominated by Quercus ilex L. in the tree layer and shrubs such as Arbutus 
unedo L., Erica arborea L., Pistacia lentiscus L., Phillyrea latifolia L., and Myrtus 
communis L. The degradation stages of the association are characterized by 
garrigues with Rubus ulmifolius Schott. and Euphorbia dendroides L. (Brullo 
et al 1977). 

The three myxomycete species collected — identified as Craterium 
aureonucleatum (Physaraceae), Perichaena quadrata (Trichiaceae), and 
Physarum mutabile (Physaraceae) — are new records for Italy. 


Materials & methods 

The investigation was carried out weekly on Pantelleria from October 2007 to May 
2009 in Mediterranean maquis and garrigues ecosystems. The collected samples were 
identified by observing sporocysts in a water solution under Leica DMLB microscope. 


466 ... Compagno, La Rosa & Venturella 


Nomenclature of the recorded taxa follows MycoBank (http://www.mycobank.org/). The 
macro- and microscopic features of peridium, stipe (if present), hypothallus, capillitium, 
columella and/or pseudocolumella, and spores were observed. The habitat type was 
noted for each recorded species. Voucher specimens were deposited in Herbarium 
Mediterraneum Panormitanum, Palermo (PAL). 


Species recorded 


Craterium aureonucleatum Nann.-Bremek. FIG. 1 


Teh Le: 


yam, =£=> a> 
(¢- 3; ay ~f . . 
( | Ss 
\ _* = se 

“= = 
" = 
~ = 4 

Ss b 
- - —— 


Fic. 1: Craterium aureonucleatum. [PAL 001(Myz)] 
a. Sporocysts. b. Spores. c. A net with yellow nodes in the capillitium. 


Myxomycetes new to Italy ... 467 


Sporocysts with a short stem, grouped, sub-conical, 0.3-0.5 mm in diameter 
and height, orange-dull brown. Hypothallus like a wide membranous disk, 
ridged, orange, stem not exceeding 0.2 mm in height. Peridium with two 
appressed layers, translucent. The outer layer of peridium is rather uniform 
while the inner layer is membranous. ‘The two layers are orange at the base, 
colorless or yellow in the upper part. The dehiscence of the peridial apex is 
circular, forming a cup-shaped structure. The capillitium consists of a net with 
small meshes, pale yellow with numerous cells, often branched, calcareous 
yellow knots, pseudocolumella orange-yellow. Spore print dark brown; spores 
dark brown-lilac in transmitted light, 9-10 um diam., covered by light tiny 
warts. 

SPECIMEN EXAMINED: ITALY. SicILy, TRAPANI PROVINCE, Pantelleria, Cuddia di Mida 

and Montagna Grande, on litter of Quercus ilex L. and Erica arborea L. 10 Nov 2008, coll. 

R. Compagno [PAL 001 (Myz)]. 
Comments - The sporocyst morphology in our collected specimens differs 
slightly from the original description of Ing (1999), where sporocysts are 
described as sub-globose, not sub-conical. This difference may be due to 
ecological conditions and infraspecific variability. 

Craterium aureonucleatum has been recorded from Canary Islands, 
Germany, Netherlands, and Scotland (Beltran & al. 2004, Ing 1999, Nannenga- 
Bremekamp 1991). 


Perichaena quadrata T. Macbr. FIG. 2 


tam. .¢ 
o > ~— 
a 8 ay "4 
he aie} . 7 


77 - ror! 
north Ti ist 4 Bibi HE 


i nalitte c TL VA Gk OC 
eet sa aris 


at fs 
' 


hes 
‘gal 
3 


Fic. 2: Perichaena quadrata [PAL 002(Myz)]. a. Sporocysts. b. Spores and capillitium. 


468 ... Compagno, La Rosa & Venturella 


Sporocysts rarely single, usually appressed with polyhedral shape, discoid, 
sometimes lengthened, purple-brown or entirely black, with exudates of calcium 
oxalate, 0.1-0.5 mm in diameter. Hypothallus extended under the sporocysts, 
dark brown. Peridium double, initially adherent, the other layer thick and 
opaque, brownish with inclusion of the inner membrane and translucent, pale 
yellow, smooth. The capillitium consists of thin tubules, usually elastic and 
abundant, sometimes with rings that segment the tubules. Spore print yellow; 
spores yellow in transmitted light, 9-12 um diam., lightly punctate. 

SPECIMEN EXAMINED: ITALY. SICILY, TRAPANI PROVINCE, Pantelleria, Bugeber, on dead 

stems of Rubus ulmifolius Schott, 15 Feb 2009, coll. R. Compagno [PAL 002 (Myz)]. 
ComMENTs - Perichaena quadrata has also been recorded from Africa, Asia, 
Australasia, Europe (including France and Spain), and North America (Lado 
1994, The Eumycetozoan Project — http://slimemold.uark.edu/index.htm). 


Physarum mutabile (Rostaf.) G. Lister FIG. 3 


| 
a wy a ti 

- aT ! ert - zg 

patagbes as Bs i Lor Si ate ees 

- 


ye, . 2 e. basil os8he 
ais 4 : ar : ara ate: 
if 4 i iii er Soa - 35h, ug yee Oi i 
7 zs ree oe 
Fic. 3: Sana ree mutabile [PAL 003(Myz)]. a. Sporocysts. b. Soe 


Tei 


Sporocysts with a short stipe; sub-globose; 0.3-0.6 mm wide and 2 mm long; 
white or pale grey. Hypothallus membranous, colorless, pale yellow or pale 
brown, sometimes with exudates. If present, the stipe is short, yellow to ochre, 


Myxomycetes new to Italy... 469 


usually covered by exudates. Peridium single, thin, colorless, smooth or rarely 
with calcium carbonate flakes, upper crack irregular. Capillitium persistent, 
elastic, with small meshes and white nodules. Pseudocolumella located in the 
center of sporocyst or at the base of plasmodiocarp. Spore print black; spores 
purple-brown in transmitted light, 7-10 um diam., densely spinulose with 
spines sometimes grouped. 

SPECIMEN EXAMINED: ITALY. Sicity, TRAPANI PROVINCE, Pantelleria, Montagna 


Grande, on litter of Q. ilex L. and E. arborea L., 18 Oct 2007, coll. R. Compagno [PAL 
003 (Myz)]. 


CoMMENTS — P. mutabile has been recorded from Africa, Asia, Europe, North 
America and South America (Emoto 1977, Ing 199, Lado 1994, Nannenga- 
Bremekamp 1991, The Eumycetozoan Project — http://slimemold.uark.edu/index. 
htm). 


Conclusions 

The new records of C. aureonucleatum, P. quadrata and P. mutabile extend 
their known European distribution into Italy and add valuable information 
regarding substrata. 


Acknowledgements 

The authors wish to thank Dr Franco Bersan (Trieste, Italy) and Dr Gabriel Moreno 
(Alcala de Henares, Madrid, Spain) for critically reviewing the manuscript and Cassandra 
Funsten (USA) for her help with linguistic revision. 


Literature cited 

Beltran E, Lado C, Barrera J, Gonzales E. 2004. Myxomycetes diversity in the laurel forest of 
Garajonay National Park (Canary Islands, Spain). Systematics and Geography of Plants 74: 
159= 173: 

Brullo S, Di Martino A, Marceno C. 1977. La vegetazione di Pantelleria (studio fitosociologico). 
Pubblicazioni Istituto Botanico Universita di Catania, pp. 5-110. Tipografia Coniglione, 
Catania. 

Emoto Y. 1977. The myxomycetes of Japan. Sangyo Tosho Publishing Co., Ltd. Tokyo, Japan. 263 
p. 

Gianguzzi L. 1999. Vegetazione e bioclimatologia dell’isola di Pantelleria (Canale di Sicilia). Braun- 
Blanquetia 22: 3-70. 

Ing B. 1999. The myxomycetes of Britain and Ireland: an identification handbook. The Richmond 
Publishing Co. Ltd., England. 374 p. 

Lado C. 1994. A checklist of myxomycetes of the Mediterranean countries. Mycotaxon 52: 
117-185. 

Nannenga-Bremekamp NE. 1991. A guide to temperate myxomycetes. Biopress Limited, England. 
409 p. 


ISSN (print) 0093-4666 © 2012. Mycotaxon, Ltd. ISSN (online) 2154-8889 


MY COTAXON 


http://dx.doi.org/10.5248/120.471 
Volume 120, pp. 471-475 April-June 2012 


Tuber microsphaerosporum and Paradoxa sinensis spp. nov. 


Li FAN’, JIN-ZHONG CAO? & Yu Lr’ 


' College of Life Science, Capital Normal University, 

Xisanhuanbeilu 105, Haidian, Beijing 100048, China 
? Institute of Mycology, Jilin Agricultural University, Changchun 130118, China 
* CORRESPONDENCE TO: fanli@mail.cnu.edu.cn 


ABSTRACT — Two new truffle species from China are described and illustrated. Tuber 
microsphaerosporum is recognized by its small ascospores and 1-7-spored asci. Paradoxa 
sinensis is separated from the European P. monospora by its pale color and larger ascospores. 


KEY worps — Ascomycota, mushroom market, Tuberaceae 


Introduction 

Knowledge about truffles in China has become richer since the first 
publication on Tuber in China by Liu (Liu 1985). Of the many species found, 
a high percentage has proved endemic (Wang 1988; Chen et al. 2005, 2007; 
Fan et al. 2011, 2012a, b). Recently we obtained two truffle-like fungi from a 
mushroom market in Kunming that we recognized as two new truffle species, 
a Tuber and a Paradoxa, described and illustrated here. 


Materials & methods 

The fresh fruiting bodies were collected from a local mushroom market in Kunming, 
China. Macro- and microscopic characters were described from fresh specimens. 
Sections were cut by razor-blade and mounted in 3% KOH for observation or stained 
in Melzer’s reagent, rinsed, and mounted in polyvinyl lactic glycerol for permanent 
slides for archiving to be kept with dried specimens. For scanning electron microscopy 
(SEM), ascospores were scraped from the dried gleba onto doubled-sided tape, which 
was mounted directly on an SEM stub, coated with gold-palladium, and examined 
and photographed with a Hitachi S-4800 SEM. The specimens are deposited in BJTC 
(Herbarium Biology Department, Capital Normal University). 


Taxonomy 


Tuber microsphaerosporum L. Fan & Yu Li, sp. nov. Fic. 1 
MycoBank MB 564356 


472 ... Fan, Cao & Li 


Fic. 1. Tuber microsphaerosporum (BJTC FAN152, holotype) 
a. Ascoma. b-c. Asci and ascospores observed under light microscope. 
d. Ascospores observed under SEM. 


Differs from other Tuber species with globose ascospores by its smaller ascospores, up 
to 7 per ascus. 

Type: China. Yunnan Province, Kunming, from a mushroom market. 20 Dec. 2010, Jin- 
Zhong Cao 115 (Holotype, BJTC FAN152). 


ETYMOLOGY: microsphaerosporum (Lat.), referring to the small globose ascospores. 


ASCOMA 2 cm in diam., subglobose, soft or slightly firm, solid, surface smooth, 
poorly pubescent to nearly glabrous, yellowish white to light yellow-brown 
at maturity. Odor slight, not pungent. PERIDIUM 250-350 um thick, two 
layers; outer layer 100-150 um thick, pseudoparenchymatous, composed 
of subangular or subglobose cells mostly 12.5-30 um in diam., with thin or 
slightly thickened walls, pale yellowish, intermixed with larger swollen cells 
of 70-80 x 40-50 um with thickened walls 2.5-3 um in diam.; inner layer 
150-200 um thick, composed of intricately interwoven hyphae, thin-walled, 
branching, hyaline, septate, 3-5 um in diam. The outermost cells giving rise a 
few hyphae-like hairs, the hairs 20-50 x 2.5-3 um, septate, thin-walled, light 
brown. GLeEBA light brown at maturity, marbled with large, rarely branching 
and white veins continuous with inner peridium. Asc1 globose, subglobose or 
broadly ellipsoid, hyaline, thin-walled, 67.5-105 x 60-70 um, mostly sessile, 
sometimes with a short stalk, 1-7-spored, mostly 3-6-spored. AscosPORES 


Two new truffle species from China ... 473 


globose, brown at maturity, 17.5-27.5 um in 2-7-spored asci and 32.5-37.5 um 
in 1-spored asci excluding ornamentation; ornamentation reticulate, meshes 
regular or irregular, 3-6 sided, 2.5-3 um tall, the meshes generally 3-8 across 
the spore width. 


ComMENts — Tuber microsphaerosporum differs from the other Tuber species 
with globose ascospores mainly by its smaller ascospores and the asci containing 
1-7 ascospores; moreover, its peridium structure with large swollen cells is also 
diagnostic for this new species. 

The North American Tuber californicum Harkn. and T: sphaerosporum 
Gilkey and European T. oligospermum (Tul. & C. Tul.) Trappe and T. borchii var. 
sphaerosperma Malencon also produce perfectly globose ascospores, but all also 
produce 1-4- or 1-3-spored asci. Tuber sphaerosporum is further distinguished 
by large reticulum meshes numbering 3-4 across the spore diameter while 
T. californicum has a dark-colored mature gleba and T: oligospermum has a one- 
layer peridium. 

Although we found no 8-spored asci in our holotype, 7-spored asci are not 
uncommon, it is possible that 8-spored asci may occur in the new species. 


Paradoxa sinensis L. Fan & J.Z. Cao, sp. nov. Fic. 2 
MycoBank MB 564357 


Differs from Paradoxa monospora by its yellowish to yellow-brown ascomata and larger 
ascospores. 


Type: China. Yunnan Province, Kunming, from the local mushroom market. 20 Dec. 
2010, Jin-Zhong Cao 113 (Holotype, BJTC FAN150). 


EryMo_oey: sinensis (Lat.), referring to the presumed host country. 
ASCOMA 2.5 cm in diam., subglobose, firm, solid, surface smooth, glabrous, 


yellowish to yellow-brown at maturity. Odor slight, not pungent. PERIDIUM 
200-250 um thick, one layer, prosenchymatous, composed of intricately 


Fic. 2. Paradoxa sinensis (BJTC FAN150, holotype) 
a. Asci and ascospores observed under light microscope. b. Ascoma. 


474 ... Fan, Cao & Li 


interwoven hyphae, branching, septate, the hyphae are light yellow-brown, 
slightly thick-walled and 5-7.5(-10) um broad towards the outer side of 
peridium, and hyaline, thin-walled and 5-7.5 um broad towards the inner 
side. GLEBA blackish at maturity, marbled with large, rarely branching and 
greyish or grey-brown veins continuous with the peridium. Asc1 1-spored, 
rarely 2-spored, globose or subglobose for 1-spored and long-ellipsoid for 
2-spored, 77.5-92.5 x 80-85 um for 1-spored and 120 x 80 um for 2-spored, 
hyaline, thick-walled, 5-7.5 um thick, sessile or with a short tail. AscosPORES 
globose, red-brown to dark red-brown at maturity, (55-)62.5-72.5 um in diam. 
excluding ornamentation, ornamentation reticulate, irregular, meshes varying 
greatly in shape and size, 5-20 um in diam., 1 um high, averagely 6-10 across 
the spore width. 


ComMENTS — Paradoxa sinensis closely resembles the European P monospora 
Mattir., but Ceruti (1960) describes the ascomata of P monospora as black 
brown and the ascospores as somewhat smaller (50-60um in diam.). The third 
Paradoxa species, P. gigantospora (Y. Wang & Z.P. Li) Y. Wang (Wang & Hu 
2008), was originally described from China by Wang & Li (1991, as Tuber 
gigantosporum) and is diagnosed by its large elliptic ascospores (<100 um). 

Paradoxa, originally described from Italy in 1935, was monotypic until Wang 
& Hu (2008) transferred T. gigantospora to it. Paradoxa sinensis represents 
the third species in the genus, which differs from Tuber mainly by its single- 
spored asci. Vizzini (2008), who treated Paradoxa as a synonym of Tuber, 
transferred P. monospora to Tuber as T:! monosporum. Kinoshita et al. (2011) 
seemingly accepted this classification when they listed Paradoxa gigantospora 
as a synonym of Tuber gigantosporum. However, in all three Paradoxa species, 
the dominant character of one larger ascospore per ascus is always connected 
with peculiar reticulum on the spore surface, which is highly irregular and 
greatly lower, suggesting a taxonomic difference between Paradoxa and most 
typical Tuber species. More research is needed to elucidate whether the two 
genera are independent or conspecific. We prefer to treat Paradoxa and Tuber 
as independent genera for the time being. 


Acknowledgments 

We are grateful to Prof. Zhu-Liang Yang (Kunming Institute of Botany, Chinese 
Academy of Sciences) and Prof. Anthony Whalley (School of Pharmacy and 
Biotechnology, Liverpool John Moores University) for reviewing the manuscript. 
The study was supported by the National Natural Science Foundation of China (No. 
30770005, 30870008) and the Beijing Natural Science Foundation (No. 5072006). 


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spinose-reticulate spored Tuber species. Mycotaxon 94: 1-6. 

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ISSN (print) 0093-4666 © 2012. Mycotaxon, Ltd. ISSN (online) 2154-8889 


MYCOTAXON 


http://dx.doi.org/10.5248/120.477 
Volume 120, pp. 477-492 April-June 2012 


Entomophthoromycota: a new phylum and 
reclassification for entomophthoroid fungi 


RICHARD A. HUMBER 


USDA-ARS Biological Integrated Pest Management Research, 
Robert W. Holley Center for Agriculture and Health, 
Tower Road, Ithaca, New York 14853-2901, U.S.A. 


CORRESPONDENCE TO: richard. humber@ars.usda.gov 


ABSTRACT — One result of the recent phylogenetically based rejection of the phylum 
Zygomycota was the description of the subphylum Entomophthoromycotina (not assigned to 
any phylum) for fungi traditionally treated in the order Entomophthorales. More extensive 
gene-based analyses of these fungi suggest that they represent a monophyletic lineage 
distinct from all other fungi that deserves now to be recognized at the level of a new fungal 
phylum. These molecular data and further analyses of more traditional taxonomic criteria 
lead to this reclassification that still treats these fungi in six families but recognizes the new 
classes Basidiobolomycetes, Neozygitomycetes, and Entomophthoromycetes as well as the new 
order Neozygitales. Ballocephala and Zygnemomyces are excluded from Entomophthorales 
(Meristacraceae) and should be reclassified among the Kickxellomycotina. 


KEY worps — Zygomycetes, sexuality, homothallism 


Introduction 

The reclassification of fungi by Hibbett et al. (2007) as the complement 
to a kaleidoscopic phylogenetic study all major fungal groups (James et al. 
2006) validated the long-accepted sense that Zygomycota was polyphyletic, 
and recognized five taxa to replace this phylum: The phylum Glomeromycota 
accommodates arbuscular mycorrhizal fungi, and all other zygomycetes were 
distributed among subphyla Entomophthoromycotina, Kickxellomycotina, 
Mucoromycotina, and Zoopagomycotina without assignment to any phylum. It 
was assumed that subsequent research would determine whether any of these 
subphyla should be regrouped as part of an effort that would necessarily result 
in the recognition of one to four new phyla for these fungi. 

The major characters traditionally used to classify the Entomophthorales 
have been thoroughly reviewed (summarized in Humber 1975, 1981, 1982, 


478 ... Humber 


1984) and were applied to six families in the last major reclassification of 
Entomophthorales (Humber 1989); this taxonomy is widely accepted despite 
a few minor differences in the treatments of some entomopathogenic genera 
(Balazy 1993; Keller 1987, 1991, 1997; Keller & Petrini 2005). Until now, 
however, there have not been phylogenetic studies on a sufficiently broad range 
of their genes and taxa to propose a more contemporary revision. 

The fungi in Entomophthoromycotina pose a few mycological puzzles 
(discussed below) for refining their current classification: Both the ultrastructure 
of the mitosis-associated organelle and early phylogenetic studies suggested that 
Basidiobolus has affinities with chytrid fungi and might better be excluded from 
Entomophthorales. Further, significant gaps in the gene-based understandings 
of entomophthoroid fungi exist because many taxa are very rarely collected 
and/or resist growth in vitro. Among these understudied taxa, Neozygites and 
related species represent the largest and most important ‘black boxes’ for which 
needed data remain unavailable. 

The gene sequences now conceded to have taxonomic value for many 
fungi (nuclear rDNA genes, translation-elongation factor, $-tubulin, etc.) 
have been used in studies of a few entomophthoraleans in a more diverse set 
of fungi (Nagahama et al. 1995, Jensen et al. 2001, White et al. 2006) and for 
narrower studies of entomophthoralean species or species complexes (Jensen & 
Eilenberg 2001, Nielsen et al. 2001). A markedly different molecular approach 
comparing amino acid sequences for proteins (including some of the same 
proteins whose DNA sequences are widely used) has placed Entomophthorales 
outside of the true fungi (Einax & Voigt 2004, Liu & Voigt 2010, Voigt & Kirk 
2011). While some skepticism about the meaning of such results based on 
amino acid sequences must be maintained, these findings do demonstrate 
the existence of distinct differences between all other groups of zygomycete 
fungi and Entomophthoromycotina. The amino-acid sequence-based ‘exclusion’ 
of Entomophthorales from the fungi echoes the hypotheses about placing 
Basidiobolus with watermolds but such ‘anomalous’ conclusions also suggest 
that molecular analyses based on highly limited inputs can yield results that 
have little sensibility from the broader perspective of the overall biology of the 
organisms analyzed. 

A series of phylogenetic studies on entomophthoraleans (being prepared 
by A. Gryganskyi, R. Vilgalys, R. Humber, and other authors) incorporated 
more genes and a much broader range of entomophthoroid taxa than any 
earlier studies. These new analyses confirm the finding by James et al. (2006) 
that entomophthoroid fungi are a monophyletic group and that this group 
does include Basidiobolus and Basidiobolaceae. A reasonable integration of 
all results of traditional and phylogenetic analyses of entomophthoroid fungi 
suggests that they are distinct from all other fungi (including those in the other 


Entomophthoromycota phyl. nov. ... 479 


unaffiliated zygomycete subphyla; Hibbett et al. 2007) and may occupy the 
most basal position among all non-flagellate fungi. The best-supported, most 
appropriate conclusion about the status of these fungi is, therefore, to recognize 
them as a new phylum in kingdom Fungi. 


Materials & methods 

Cultures and specimens used for the analyses discussed here are primarily from the 
USDA-ARS Collection of Entomopathogenic Fungal Cultures (ARSEF; http://www.ars. 
usda.gov/Main/docs.htm?docid=12125) and its associated herbarium. Unpublished 
molecular results of analyses of various genes are based on sequences of cultures obtained 
from ARSEF or isolated from nature and, in most cases, subsequently deposited in 
ARSEF. Other results involving reports on fungi and analyses using other isolates and 
specimens were completed at the Zoology Section of the Department of Agriculture and 
Ecology, University of Copenhagen, or at Agroscope FAL Reckenholz Eidgenéssische 
Forschungsanstalt fir Agrardkologie und Landbau (Ziirich). 


Major taxonomic issues affecting this reclassification 


‘Linkage’ of Basidiobolus with flagellate fungi 

The nuclei of entomophthoralean fungi and the details of their mitoses 
present a comparatively richer number and variety of characters than in most 
other fungal groups, and these nuclear characters are taxonomically informative, 
especially above the generic rank (Humber 1975, 1981, 1982, 1984, 1989). 
Mitosis by the huge nuclei of Basidiobolus species is sufficiently unusual to 
have been studied repeatedly (Olive 1907, Robinow 1963, Sun & Bowen 1972, 
Tanaka 1978). Among other surprises, the location of the mitosis-associated 
organelle in this genus is not fixed at the spindle poles and can even occur in the 
plane of the metaphase plate (Sun & Bowen 1972). The real controversies about 
this mitosis arose, however, when this organelle proved to be a short cylinder 
with a ring of 11-12 singlet microtubules (McKerracher & Heath 1985), and 
comparisons between this structure and centrioles were used to question the 
phylogenetic placement of this fungus; Basidiobolus remains the ONLY organism 
producing no flagellum in its life history for which microtubules are proven to 
be present in a mitosis-associated organelle. Nonetheless, any hypothesis that 
this organelle in Basidiobolus (but whose existence, location and ultrastructure 
remain unconfirmed from other fungi in its family) is linked with or derived 
from centrioles seems neither credible nor responsible: The 9x3 microtubular 
arrangement in centrioles and kinetosomes is invariant among ALL phyla of 
eukaryotes having flagella; conversely, no organelle with microtubules arranged 
like those in Basidiobolus is known from any other organism. 

Later findings of gene sequence similarities between Basidiobolus and 
several chytrid and blastocladian watermolds (Nagahama et al. 1995, Jensen et 
al. 1998, Tanabe et al. 2000, White et al. 2006) have also been used to suggest 


480 ... Humber 


that Basidiobolus might not belong in Entomophthorales. However, other studies 
allied Basidiobolus with kickxelloid fungi from Harpellales (Keeling 2003, 
Tanabe et al. 2004) and one placed Conidiobolus coronatus (whose inclusion in 
Entomophthorales was never disputed) on a branch with the blastocladialean 
genus Allomyces (Tanabe et al 2005). These divergent findings underscore the 
peril of suggesting phylogenetic relationships among major fungal groups after 
comparing very limited sequence data and very sparse samplings of taxa within 
large and inherently diverse groups of fungi. 

The recent survey of phylogenetic relationships within kingdom Fungi 
(James et al. 2006) supported the continued placement of Basidiobolus in 
Entomophthorales. Regardless of molecular or ultrastructural rationales to the 
contrary, hypotheses that Basidiobolus is not entomophthoroid are nonsensical 
if one considers the overall biology of these fungi. Basidiobolus and its relatives 
share many novel features in common with other entomophthoroid taxa 
despite the scant few pieces of data suggesting otherwise. It is necessary to 
bring a broader perspective to the uncertainties about Basidiobolus: Despite 
any and all evidence to the contrary, if this genus does not belong among 
the entomophthoroid fungi, then jusT WHERE AMONG FLAGELLATE (OR ANY 
OTHER) FUNGI SHOULD IT BE CLASSIFIED? The lack of any comparably well- 
supported answers for this question should quash any residual doubts about 
where Basidiobolus belongs. 


Neozygitaceae: a special ‘problem’ in data gathering 

The status of taxa in Neozygitaceae also presents a (temporary) problem 
for reclassifying the Entomophthorales. Very few cultures of Neozygites species 
are available in vitro, and, sadly, all current cultures of Neozygites are of mite 
pathogens with rough-walled, nearly globose zygospores. No gene-based data 
are available for N. fresenii [=N. aphidis, the type species] or other hemipteran 
pathogens that form ovoid, smooth-walled resting spores. A further taxonomic 
frustration is that these morphological and host differences suggest that 
Neozygites may eventually be split into two or more genera, but molecular 
data will be required to support such a decision. DNA-based evaluations of 
fungi from Neozygitaceae is encumbered by difficulties experienced in multiple 
laboratories to obtain clean DNA useful for amplifying and sequencing 
the genes needed to determine their phylogenetic relationships. While the 
recognition of Neozygitomycetes as a new class without supporting gene-based 
evidence may be dismissed by some as premature, such a treatment is the 
most reasonable for these fungi at this time, based on their known organismal 
biology (that integrates and represents a vastly larger proportion of the gnome 
than those few genes now so widely treated as sufficient to complete high-level, 
phylogenetically sound reclassifications of virtually all other fungal groups). 


Entomophthoromycota phy]. nov. ... 481 


Distinct differences between neozygitoid fungi and either basidioboloid or 
entomophthoroid taxa supports the description of three classes in this new 
phylum: As in basidioboloid fungi, neozygitoid fungi exert strong control 
in vegetative cells over nuclear number (usually four in Neozygites), have a 
central mitotic metaphase plate (Butt & Humber 1989), and, perhaps most 
significantly, a round of mitosis in gametangial cells precedes conjugation and 
zygosporogenesis while only one nucleus from each gametangium enters each 
zygospore (Keller 1997). As in entomophthoroid fungi, all neozygitoid taxa 
are obligate pathogens of insects or mites, and the nuclear membrane remains 
intact throughout mitosis. On the other hand, the chromosomes of neozygitoid 
fungi differ from basidioboloid and entomophthoroid fungi in being vermiform 
and of moderate size, condensing during mitosis but uncoiled (euchromatic) 
during interphase. Neozygites mitoses (Butt & Humber 1989) resemble those 
in animal or plant cells more closely than those in any other entomophthoroid 
fungi. 

The presence of many novel characteristics shared among all of the fungi 
traditionally classified in order Entomophthorales underscores the need to keep 
these fungi together in a phylogenetically supported, coherent group and to 
pursue further studies to obtain more vital data about the genes, development, 
pathobiology, and other aspects for a better understanding of these fungi that 
can be very important naturally occurring biological control agents. Because 
these fungi occupy a very ancient position of the fungal tree of life it is also 
important to note that a better understanding should help to understand 
more about the enigmatic transition of fungi (as also occurred with plants and 
animals) from waterborne to terrestrial life forms. 


Taxonomy 


Entomophthoromycota Humber, phyl. nov. [TABLE 1] 
MycoBank MB 564375 

VEGETATIVE GROWTH AS hyphae, hyphal bodies, or yeast-like; cells broad, walled or 
protoplastic. CONIDIOPHORES simple or digitate, each branch forming one conidiogenous 
cell and one conidium. PRIMARY SPORES conidia, uni- to multinucleate, usually forcibly 
discharged; usually forming one or more types of SECONDARY CONIDIA. RESTING SPORES 
homothallic zygospores or azygospores. HaBit mostly as arthropod pathogens, but 
some saprobes or specialized phytopathogens. 


TyPE GENUS: Entomophthora Fresen. 1856. 


CONIDIOPHORES rise from mycelium or from body of host, usually with positive 
phototropic orientation, simple or apically (digitately) branched, with single 
conidiogenous cell on each branch giving rise to a single conidium or simple 
erect conidiophore becomes septate and each cell forms a single conidium. 
PRIMARY CONIDIA (not sporangia) with wall layers continuous with those on 


482 ... Humber 


TABLE 1. Proposed new classification for Entomophthoromycota. 
New taxa described here are listed in boldface italics. 


Puyitum Entomophthoromycota Humber, phyl. nov. 
Cass Basidiobolomycetes Humber, cl. nov. 
ORDER Basidiobolales Caval.-Sm., Biol. Rev. 73: 246. 1998. 
FamILy Basidiobolaceae Claussen, Syllab. Pflanzenfam., Edn 9 & 10: 45. 1924. 
Basidiobolus Eidam, Beitr. Biol. Pflanz. 4: 194. 1886. 
Other new, undescribed genera (R.A. Humber, B. 
Huang & K. Hodge, unpublished). 


Ciass Neozygitomycetes Humber, cl. nov. 
ORDER Neozygitales Humber, ord. nov. 
FamiLy Neozygitaceae Ben-Zeev, R.G. Kenneth & 
Uziel, Mycotaxon 28: 321. 1987. 
Apterivorax S. Keller, Sydowia 57: 47. 2005. 
Neozygites Witlaczil, Arch. Mikr. Anat. 24: 601. 1885. 
Thaxterosporium Ben-Zeev & R.G. Kenneth, Mycotaxon 28: 323. 1987. 


Cxass Entomophthoromycetes Humber, cl. nov. 
ORDER Entomophthorales G. Winter, Rabenh. Krypt.-Fl., Edn 2, 1(1): 74. 1880. 
FaMILy Ancylistaceae J. Schrot., Nat. Pflanzenfam. 1(1): 92. 1893. 
Ancylistes Pfitzer, Monatsb. Konigl. Preuss. Akad. Wiss. Berlin: 396. 1872. 
Conidiobolus Bref., Untersuch. Gesammtgeb. Mykol. 6: 37. 1884. 
Macrobiotophthora Reukauf, Centrabl. Bakt., Abt 1, 63: 390. 1912. 
FamILy Completoriaceae Humber, Mycotaxon 34: 453. 1989. 
Completoria Lohde, Tagebl. Versamml. Deutsch. Naturf. Aertze 47: 206. 1874. 
FamiLy Entomophthoraceae Nowak., Bot. Ztg. 35: 35. 1877. 
SUBFAMILY Entomophthoroideae S. Keller, Sydowia 57: 28. 2005. 
Batkoa Humber, Mycotaxon 34: 446. 1989. 
Entomophaga A. Batko, Bull. Polon. Acad. Sci. Sér. Biol. Sci. 12: 325. 1964. 
Entomophthora Fresen., Bot. Ztg. 14: 883. 1856. 
Eryniopsis Humber, Mycotaxon 21: 258. 1984, pro parte. 
Massospora Peck, Rep. New York State Mus. 31: 44. 1879. 
SUBFAMILY Erynioideae S. Keller, Sydowia 57: 33. 2005. 
Erynia (Nowak. ex A. Batko) Remaud. & 
Hennebert, Mycotaxon 11: 333. 1980. 
Eryniopsis Humber, Mycotaxon 21: 258. 1984, pro parte. 
Furia (Batko) Humber, Mycotaxon 34: 450. 1989. 
Orthomyces Steinkraus, Humber & J.B. Oliv., J. Invertebr. Pathol. 72: 5. 1998. 
Pandora Humber, Mycotaxon 34: 451. 1989. 
Strongwellsea A. Batko & Weiser, J. Invertebr. Pathol. 7: 463. 1965. 
Zoophthora A. Batko, Bull. Polon. Acad. Sci. Sér. Biol. Sci. 12: 323. 1964. 
FamILy Meristacraceae Humber, Mycotaxon 34: 456. 1989. 
Meristacrum Drechsler, J. Wash. Acad. Sci. 30: 250. 1940. 
Tabanomyces Couch, RV Andrejeva, Laird & Nolan, 
Proc. Natl. Acad. Sci. USA 76: 2302. 1979. 


Entomophthoromycota phy]. nov. ... 483 


conidiogenous cells, inner wall layer invaginating to form two-layered septum 
between conidium and conidiogenous cell; almost always forcibly discharged 
(several possible mechanisms are known). SECONDARY CONIDIA formed by 
most taxa: if forcibly discharged from short secondary conidiophore then 
usually similar in shape to primary conidium; if passively dispersed from long, 
thin (capillary) secondary conidiophore then usually distinctly differing in 
morphology from primary conidium. RESTING sPpoRES (when mature) with 
thick, distinctly 2-layered walls, colored or hyaline, outer layer surface smooth 
or variously decorated; formed as zygospores (after gametangial conjugation) 
or azygospores (with no conjugation) either in the axis of the parental cells 
or budded off laterally; nuclear number in mature spores varies from 2 (from 
initiation or progressively reducing to 2) to multiple; germinating directly 
by forming germ conidiophore and germ conidium (usually resembling a 
secondary spore type) or indirectly by forming a small germ mycelium and 
then germ conidia (usually like primary conidia). HasiTs: saprobes in soil or 
litter, primary pathogens of arthropods (insect, mites, spiders) or other soil 
invertebrates (nematodes, tardigrades), or highly specific phytopathogens 
(e.g., of desmid algae or fern gametophytes). ARTHROPOD PATHOGENS may 
form specialized organs: RHIZOIDs with or without differentiated holdfasts may 
anchor host to substrate, and cysTip1A may perforate host cuticle and facilitate 
emergence of conidiophores. 


Primary and secondary conidia are the major spore forms in this phylum and 
constitute the primary basis for the taxonomy of these fungi. The resting spores 
are formed much less commonly than are conidia. ‘The majority of species 
are pathogens of arthropods although pathogens of other soil invertebrates 
(nematodes and tardigrades) or of plants (desmid algae or fern gametophytes) 
are rare. The primary habit (especially in Basidiobolaceae and Ancylistaceae) 
may be in soil and plant detritus, but some species in these groups are best 
known as colonists of amphibian and reptile guts (Basidiobolus) or as facultative 
or obligate entomopathogens (Conidiobolus). 

Any continued use of subphylum Entomophthoromycotina Humber 
(Hibbett et al. 2007: 517) is now superfluous until any future decision divides 
Entomophthoromycota into subphyla. This reclassification does not take up 
the phylum Basidiobolomycota Doweld (2001; LXXVII) because Doweld’s 
name was proposed as part of a general reclassification of all fungi that does 
not agree with current understandings of fungal biology and relationships 
and, as circumscribed, Basidiobolomycota and the class Basidiobolomycetes 
Doweld (2001: LXXVII) used fragmentary knowledge of characters that may 
not apply to all taxa intended to be included while failing to account in any 
way for most taxa specifically included in this circumscription of phylum 
Entomophthoromycota; also see discussion below for class Basidiobolomycetes. 


484 ... Humber 


Basidiobolomycetes Humber, cl. nov. 

MycoBank MB 564376 
Differs from Entomophthoromycetes and Neozygitomycetes by unusually large nuclei 
(often 210 um long) with a large central nucleolus that is the major feature of uninucleate 
cells. Mitoses involve barrel-shaped spindles, mitotic organelles incorporating 
microtubules (but not centrioles) but not always located at the spindle poles, and the 
nuclear content isolated from the cytoplasm by a layer of nuclear and cytoplasmic 
membrane fragments. 


Type GENUS: Basidiobolus Eidam 1886. 


VEGETATIVE CELLS uninucleate, as regularly septate mycelium or yeast-like cells 
cleaved from contents of a parental cells (e.g., so-called ‘Darmform’ growth). 
Mitosis begins with fragmentation of nuclear membrane and aggregation of 
these and other membranes around a nuclear zone; chromosomes numerous, 
tiny, condensed and aligned on central metaphase plate (usually embedded 
inside the nucleolus) in association with a barrel-shaped spindle, chromosomes 
uncoil during interphase. CONIDIOGENOUS CELL (CONIDIOPHORE) simple 
but with bulbous apical swelling immediately below developing conidium. 
CONIDIA uninucleate, globose, with small conical basal papilla (projecting 
into spore body but everting during discharge), unitunicate (wall layers not 
separable). CONIDIA DISCHARGE forcibly by rocket-like ejection when central 
circumscissile weakness of the subconidial swelling ruptures; the upper portion 
of the swelling discharges together with conidium but may detach during 
flight. SECONDARY CONIDIA (if formed) usually elongate, often curved, with 
or without an apical mucoid droplet, formed apically on an elongated capillary 
conidiophore, passively dispersed. RESTING spoREs (RS) usually zygospores, 
formed homothallically in axis of parental cells; gametangial nuclei undergo 
mitosis before conjugation but only one nucleus from each cell enters the 
zygospore. MATURE ZYGOSPORES have thick, bi-layered walls; RS GERMINATE 
by direct formation of germ conidium (usually a secondary conidial type: 
elongate, passively dispersed from a capillary conidiophore). 


The foremost diagnostic character for basidioboloid fungi is their huge nucleus 
(often =10 um in length) with a prominent central nucleolus that is the major 
feature of uninucleate cells (either as a broad, septate mycelium or cells cleaving 
internally in yeast-like growth mode). There is no staining of interphasic nuclei 
(nor, in any obvious manner, of mitotic chromosomes) in Basidiobolomycetes 
in aceto-orcein or other nuclear stains. The individual volumes of these nuclei 
may be many times greater than the entire cells of most ascomycete yeasts), 
and their mitoses are unusual for more than just the microtubular nucleus- 
associated organelle (McKerracher & Heath 1985): As mitosis begins, the 
nuclear envelope breaks down but the endoplasmic reticulum and other 
membranous cell components cluster around the nuclear zone so that the 


Entomophthoromycota phyl. nov. ... 485 


spindle and chromosomes remain well isolated from the cytoplasm despite the 
fragmentation of the nuclear envelope; the corollary effect of this membrane 
organization is that mitotic nuclei ‘disappear’ when viewed with light microscopy 
(Robinow 1963). 

Zygosporogenesis in Basidiobolus (Eidam 1886) is also very distinctive as 
short beak-like, lateral projections form at the septum between gametangial 
cells; gametangial nuclei move into the beaks, undergo mitosis, and the 
(uninucleate) beak cells are walled off before the septum dissolves and 
zygosporogenesis proceeds; remnants of these ‘beaks’ often remain visible on 
mature zygospores. 

As noted in the discussion for the new phylum, two available names for 
this new class were not adopted: Bolomycetes Cav.-Smith (Cavalier-Smith 1998: 
243) was based mainly on the microtubular mitotic organelle and ‘beaked’ 
zygospores in Basidiobolus. This mitotic organelle is not confirmed as present 
in all taxa in the Basidiobolaceae (including at least two still undescribed new 
genera), and the zygospores of some basidiobolaceous fungi are not “beaked’ as 
in Basidiobolus. Basidiobolomycetes Doweld (2001: LXXVII) was proposed as a 
nomen novum for Bolomycetes and cited Cavalier-Smith’s description for this 
class; Doweld neither placed nor mentioned other entomophthoralean fungi in 
any rank in his general reclassification of fungi. 


Neozygitomycetes Humber, cl. nov. 
MycoBank MB 564377 


Differs from Basidiobolomycetes and Entomophthoromycetes by vermiform, moderately 
sized chromosomes that condense during mitosis on a central metaphase plate but 
uncoil during interphase. Nuclear numbers in vegetative cells and conidia are low and 
apparently controlled at (3)-4-(5). 


TyPE GENUS: Neozygites Witlaczil 1885. 


VEGETATIVE CELLS are rod-like hyphal bodies, walled or protoplastic, usually 
with 4 (3-5) nuclei, elongating until + synchronous mitosis; daughter cells 
separate by splitting of septum. NUCLEAR NUMBER in all cell types strongly 
regulated; usually 4 (3-5) in vegetative cells and conidia, 2 in resting spores. 
MiTOsEs intranuclear, + synchronous in any cell; nuclei fusoid at metaphase 
with central, fusoid spindle; no nucleus-associated mitotic organelle observed; 
chromosomes uncoil (euchromatic) during interphase. CONIDIOPHORES 
simple; forming apical conidiogenous cell and one conidium. PRIMARY 
conip1A subglobose to broadly ovoid, basal papilla short, comparatively flat; 
forcibly discharged to short distance by papillar eversion. SECONDARY CONIDIA 
usually form quickly after primary conidial discharge, most commonly form as 
capilliconidia (that are the primary infective units). RESTING spoREs bud from 
short conjugation bridge between rounded-up hyphal bodies (gametangia) 


486 ... Humber 


after preconjugational mitosis in contacting gametangia; zygospore receives 
one nucleus from each gametangium; only outer wall layer is melanized. 
Mature resting spores with two adjacent round fenestrae (‘holes’ through outer 
wall layer) and raised ridge of gametangial wall remnants between them. 


Melanization of all spore types is a major feature of Neozygitomycetes. Primary 
and secondary conidia are pale, smoky gray; individual resting spores are much 
more strongly colored, and dark gray to black in mass. 


Neozygitales Humber, ord. nov. 
MycoBank MB 
ORDER having all characteristics of class Neozygitomycetes. 


TYPE GENUS: Neozygites Witlaczil 1885. 


This order has all characters of class Neozygitomycetes (which includes only a 
single order and family). 


Entomophthoromycetes Humber, cl. nov. 
MycoBank MB 564381 


Differs from Basidiobolomycetes by lack of uniformly uninucleate cells, nuclear 
morphology, details of mitoses, and modes of zygosporogenesis; and from 
Neozygitomycetes by cells not having uniformly small numbers of nuclei, details of 
mitoses, and lack of melanization of all spore types. 


TYPE GENUS: Entomophthora Fresen. 1856. 


VEGETATIVE GROWTH as coenocytic mycelium or rod-like to variably shaped 
hyphal bodies, walled or naturally protoplastic; if wall-less, rod-like to highly 
variable in shape and/amoeboid. CONIDIOPHORES simple or digitately branched, 
each branch with a single apical conidiogenous cell, or (in Meristacraceae) an 
unbranched erect, septate conidiophore forming one conidium per cell. Conidia 
unitunicate (wall layers not separating in liquid) or bitunicate (with separable 
outer wall layer); variously shaped, uni- to multinucleate, with basal papilla flat, 
conical or rounded; forcibly discharged by papillar eversion in most genera. 
SECONDARY CONIDIA more or less similar in shape to primary conidia and 
forcibly discharged if formed on short secondary conidiophore, or elongate and 
passively dispersed if formed on elongated capillary secondary conidiophore. 
NUCLEI (interphase) with small nucleolus, interphasic heterochromatin present 
in Entomophthoraceae but absent in all other families; mitoses intranuclear, 
with small lateral metaphase plate lateral; interphasic chromosomes are partly 
condensed (heterochromatic) and stain readily in Entomophthoraceae but 
euchromatic (uncoiled and nonstaining) in other families. RESTING SPORES 
globose to subglobose, formed as zygospores or azygospores. HasiT obligately 
pathogenic for invertebrates (Entomophthoraceae, Meristacraceae, some 
Ancylistaceae), saprobic (some Ancylistaceae), or phytopathogenic (Completoria 
[Completoriaceae| and Ancylistes [Ancylistaceae]). 


Entomophthoromycota phyl. nov. ... 487 


This class includes all members of Ancylistaceae, Entomophthoraceae, 
Completoriaceae, and Meristacraceae but omits those entomophthoralean 
taxa reassigned here to Basidiobolomycetes or Neozygitomycetes (TABLE 1) or 
removed from Entomophthoromycota as noted below. 


Genera incertae sedis: 


Eryniopsis Humber, Mycotaxon 21: 258. 1984. 
All species are in Entomophthoraceae but would appear to be a mix of taxa 
representing both subfamilies Erynioideae and Entomophthoroideae; the type 
species, E. lampyridarum, has morphological characters suggestive of both 
subfamilies and cannot be placed in either without molecular studies. 


Tarichium Cohn, Beitr. Biol. Pflanzen 1: 69. 1870. 
This form genus for species known only from resting spores apparently represents 
a mix of species attributable to Neozygitaceae (especially species pathogenic to 
mites) and Entomophthoraceae. No new species should be added to this genus; 
DNA-based studies and morphological re-evaluations should allow most species 
to be recognized as synonyms of other species or transferred to other genera in 
Entomophthoraceae and Neozygitaceae. 


Taxa inadoptata vel excludenda: 


Massosporoideae S. Keller, Sydowia 57: 44. 2005. 
This subfamily (accommodating only the genus Massospora) seems not to be 
supported by phylogenetic evidence and is treated as a synonym of subfamily 
Entomophthoroideae. 


Ballocephala Drechsler, Bull. Torrey Bot. Club 78: 199. 1951. 

Zygnemomyces K. Miura, Rep. Tottori Mycol. Inst. 10: 520. 1973. 
These two genera are excluded from Meristacraceae and reassigned to 
Kickxellomycotina Benny (Hibbett et al. 2007) based on the bifurcate septa with 
lenticular plugs in their vegetative hyphae (Saikawa 1989; Saikawa et al. 1997). 


Discussion 

The terms ‘mitospore’ and ‘meiospore’ are not used in characterizing taxa of 
Entomophthoromycota. They were originally adopted to describe ascomycete 
and basidiomycete spores, and are not applicable to entomophthoroid fungi 
because the reproductive products and life histories of entomophthoroid 
fungi are not strictly comparable with those of the Dikarya: The thin-walled 
primary conidia (the basis for entomophthoroid taxonomy) are produced 
by the vegetative cells of these fungi, usually forcibly discharged, and usually 
able to produce one or more subsequent forms of secondary conidium if 
conidia do not germinate by producing a germ tube. Entomophthoroid 
resting spores may be conventionally sexual in nature (zygospores in which 
it may be AssUMED, although not yet proven, that karyogamy and meiotic 


488 ... Humber 


divisions occur) in Basidiobolomycetes and Neozygitomycetes; for taxa in the 
Entomophthoromycetes, and especially those in Entomophthoraceae, it was 
noted that the MORPHOLOGICAL events of sexuality (the presence or absence 
of gametangial conjugations define zygo- and azygosporogenesis, respectively) 
and the GENETIC events of sexuality (karyogamy and meiosis, that presumably 
happen during resting spore germination) may be completely independent 
processes (Humber 1981, McCabe et al. 1984). Entomophthoroid fungi may be 
the only fungi in which the morphological and genetic definitions of sexuality 
(or their absences) are present in all possible permutations and without the 
routine linkage between the morphological and genetic events of sexuality that 
is taken for granted in virtually all other types of organisms. 

No zygomycetous or flagellate fungi produce spores that can accurately be 
referred to as meiospores in the sense of basidiospores and ascospores. The 
proven or presumably ‘sexual’ spores of fungi below the subkingdom Dikarya 
are thick-walled, environmentally resistant spores (zygospores, azygospores, 
resistant sporangia, etc.) that go through a quiescent dormancy before 
germinating to undergo a type of sporulation that is neither functionally nor 
developmentally comparable to the basidiospores and ascospores that are the 
direct and obligatory developmental products of the cells in which karyogamy 
and meiosis occur. 

To obtain clean DNA and good sequence data from entomophthoroid fungi 
may be more difficult than for many, much more extensively studied fungal 
groups. Part of this difficulty might involve the physical organization of the 
genome in these fungi that might lead to overlapping but divergent sequences 
for some ‘needed’ genes. Chromosomal counts for Basidiobolus (which was 
long treated as including only one species, B. ranarum) have ranged from about 
60 (Olive 1907) to several hundred (Sun & Bowen 1972) based on kinetochore 
counts in serial sections for transmission electron microscopy. These high 
numbers suggest that polyploidization events may have occurred repeatedly 
in Basidiobolus. This possibility seems to be verified by genetic studies showing 
multiple, genetically distinct allelic forms in B. ranarum for elongation- 
translation factor genes that usually occur in single copies in the genome 
(Henk & Fisher 2012). The few chromosome counts for entomophthoraceous 
entomopathogens also suggest a tendency to towards polyploidy: While the 
nature of chromosomes and mitoses may not facilitate chromosomal counts 
in Entomophthoraceae (Olive 1906, Humber 1975), the few published numbers 
in various taxa are 8, 12 (or more), 16, and 32 (see Humber 1982). No genetic 
studies like those of Henk & Fisher (2012) are available for Entomophthoraceae 
but no genetic studies of this family with techniques ranging from allozyme 
polymorphisms (Hajek et al. 1990; B. May, personal communication) to the 
latest gene sequencing efforts suggest that these fungi simultaneously harbor 


Entomophthoromycota phyl. nov. ... 489 


multiple allelic variants at single loci; there is no indication that vegetative nuclei 
of these fungi are either diploid or include heterologous sets of chromosomes. 

The interpretation of such seemingly numerous chromosomes in some 
taxa in Entomophthoromycota becomes more problematic in the absence of 
evidence suggesting that any putatively sexual reproduction in this phylum is 
heterothallic rather than strictly homothallic. No data support the invocation 
of heterothallic sexuality (even if outbreeding events were extremely rare) to 
explain Henk & Fisher's (2012) conclusions about the Basidiobolus genome. 
Except for gametangial fusions during zygosporogenesis, no cellular fusions 
(even between the naturally protoplastic vegetative cells of some of the 
pathogenic taxa) are known from cultures or natural collections of any 
entomophthoroid fungus; such fastidious behavior by these fungi precludes 
consideration of heterokaryosis and parasexuality as a mechanism to increase 
or to sustain gene flow among taxa in Entomophthoromycota. 

It is important to note again that the Entomophthorales as traditionally 
recognized (Humber 1989) is the same group reclassified here except for the 
removal of Ballocephala and Zygnemomyces to Kickxellomycotina based on the 
bifurcate, plugged septa in their vegetative hyphae (Saikawa 1989, Saikawa et al. 
1997). Despite earlier doubts about retaining Basidiobolus in Entomophthorales, 
molecular studies of more genes and a broader spectrum of entomophthoraleans 
(A. Gryganskyi, R. Vilgalys, and R. Humber, unpublished) confirm that this 
order, as historically treated, is monophyletic. These fungi exemplify yet 
another major group for which the traditional, pre-molecular classification 
has been fundamentally confirmed (although amplified and adjusted) rather 
than overturned by phylogenetic analyses. Phylogenetic techniques must not 
be allowed to override or to supplant the existing knowledge about groups 
of organisms despite the vital inputs, seductively authoritative-looking 
dendrograms, and current pre-eminence among taxonomy methodologies. 
The best role for phylogenetic techniques should be as partners with the much 
broader (and usually older) perspectives gained by a thorough understanding 
of the overall biology as the means to determine the most sensible and best 
supported organismal classifications. 


Acknowledgments 

Special thanks are due to Andrii Gryganskyi, Annette Bruun Jensen, Kathie T. Hodge, 
and Bo Huang for the molecular results that underpin this new classification, to Walter 
Gams and Rytas Vilgalys for critical reviews, and to James L. Reveal for stimulating 
discussion of nomenclatural issues. 


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NOTE ADDED IN PROOF: Since the acceptance of this article, the bibliographic 
citations for the two molecularly based papers that underpin and justify this new 
classification of entomophthoroid fungi have become available: 


Gryganskyi AP, Humber RA, Smith ME, Miadlikovska J., Wu S, Voigt K, Walther G, Anishchenko 
IM, Vilgalys R. 2012. Molecular phylogeny of the Entomophthoromycota. Mol. Phylog. Evol. 
65: 682-694. http://dx.doi.org/10.1016/j.ympev.2012.07.026 

Gryganskyi AP, Humber RA, Smith ME, Hodge K, Huang B, Voigt K, Vilgalys R. 2012. Phylogenetic 
lineages in Entomophthoromycota. Persoonia: in press. 


ISSN (print) 0093-4666 © 2012. Mycotaxon, Ltd. ISSN (online) 2154-8889 


MY COTAXON 


http://dx.doi.org/10.5248/120.493 
Volume 120, pp. 493-503 April-June 2012 


Moniliophthora aurantiaca sp. nov., 
a Polynesian species occurring in littoral forests 


BRADLEY R. Kropp?* & STEVEN ALBEE-SCOTT?” 


‘Biology Department, 5305 Old Main Hill, Utah State University, Logan, Utah 84322 USA 
?Wilbur L. Dungy Endowed Chair of the Sciences, Environmental Sciences, 

Jackson Community College, 2111 Emmons Road, Jackson, MI USA 
*CORRESPONDENCE TO: brad.kropp@usu.edu 


ABSTRACT — A new species of Moniliophthora is described from the Samoan Islands. The 
new species is characterized by its bright orange pileus and pale orange stipe and lamellae. It 
occurs commonly on woody debris in moist littoral forests and has not been found in upland 
forests. A phylogenetic analysis of nLSU and ITS sequences indicates that Moniliophthora 
aurantiaca has an affinity with the Central and South American members of the genus. 
Possible mechanisms for the dispersal of fungi from the Neotropics to the Samoan Islands 
are discussed. 


Key worps — Agaricales, American Samoa, Crinipellis, Oceania, phylogeny 


Introduction 

The mycobiota of the Samoan Islands has received very little attention. 
What work that has been done consists of an inventory of the wood decay 
fungi and plant pathogens present in American Samoa (Brooks 2004, 2006) 
along with the recent description of an Inocybe species from the island of Tau 
(Kropp & Albee-Scott 2010). Our preliminary work on Samoan fungi indicates 
that the mycoflora of the islands is potentially quite rich and that a number of 
undescribed species is present. 

An attractive, bright orange species of Moniliophthora was recently 
discovered in littoral forest on the islands of Tutuila and Tau, American Samoa. 
In spite of its distinctive appearance and the fact that it appeared to be very 
common at the time the material was collected, no name has been found for 
this fungus among the published species of the Moniliophthora/Crinipellis 
complex. In this article, we propose a new species of Moniliophthora from the 
Samoan Islands and assess its phylogenetic position relative to other members 
of Moniliophthora and to members of Crinipellis and Chaetocalathus. 


494 ... Kropp & Albee-Scott 


Materials & methods 

Specimens were photographed in the field on natural substrates or using a gray 
card as a background and field notes were taken while the material was still fresh. The 
collections were then dried for herbarium specimens. Microscopic study of the dried 
specimens was carried out in the laboratory using a light microscope after rehydrating 
tissue sections in Melzer’s reagent. Microscopic characters were measured using oil 
immersion at 1000x except for pileus hairs, which because of their length were made 
at lower magnifications. All microscopical characters were illustrated with the aid of 
a drawing tube. Spore measurements are given as an average with ranges, whereas 
measurements of the other cells are given as ranges. Color notations were taken from 
the digital photographs using the Munsell Soil Color Charts (Munsell Color 2000) and 
Farver i Farver (Wanscher & Kornerup 1991). Formats for the Munsell and Farver i 
Farver color notations are 5YR 8/8 and 54A respectively. All specimens examined, 
including the holotype, have been accessioned into the Intermountain Herbarium 
(UTC) at Utah State University. 

DNA was extracted from dried herbarium material using standard protocols adapted 
for use in our lab (Kropp et al. 1996). Amplified PCR products were obtained using 
standard PCR protocols (White et al. 1990) for the internal transcribed spacer (ITS) 
using primers ITS4 and ITS5 (White et al. 1990) and for the nuclear large ribosomal 
subunit (nLSU) between primers LROR and LR5 (Moncalvo et al. 2000). Direct 
sequencing of the purified PCR products was done for both the nLSU and the ITS using 
the amplification primers. Both sequences for M. aurantiaca were deposited in GenBank 
(ITS = JN692482, nLSU = JN692483). 

Twenty-one species of Crinipellis representing Europe, Australasia, and North and 
South America were sampled. Taxon sampling also included four Moniliophthora species 
from Australasia, North America, and the American tropics. To identify apomorphies, 
members of the closely related Chaetocalathus and representatives of the less closely 
related Tetrapyrgos and Marasmius were used as outgroups in our analysis (TABLE I). 

Clustal X (Thompson et al. 1997) was used to align the nLSU and ITS sequences 
after they were concatenated in the phylogenetically uninformative terminal ends. 
MrBayes 3.1 was used to analyze the aligned data set, 1613 bases in length (Ronquist & 
Huelsenbeck 2003). Treespace was searched using a time reversible model of evolution 
(Maddison 1994, Rodriguez et al. 1990) and a discrete gamma distribution with six 
substitution types and some invariant sites (GTR+G+I). Every hundred trees were 
sampled from the Bayesian simulation to approximate posterior probabilities. The 
trees were simulated using the Markov Chain Monte Carlo Method (MCMC) and all 
Bayesian simulations were conducted with eight active MCMC chains, heated at 0.2, and 
started with a randomly chosen neighbor-joining tree. The first MCMC run was iterated 
for 1,000,000 generations and three subsequent MCMC simulations were done using 
1,000,000 generations, sampling every one hundredth tree. A majority consensus tree 
was calculated from the last 7000 sampled trees from a 10,000 tree data set using all runs 
to recover the posterior probabilities of the internal nodes using the sumT command in 
MrBayes. All four consensus trees recovered from the four Bayesian simulations were 
phylogenetically identical. The resulting statistics showed that the two independent 
MCMC chains converged with a standard deviation <0.001 (Ronquist & Huelsenbeck 


TABLE 1. Species, geographic origins, and GenBank accession numbers 


TAXON 

Chaetocalathus columellifer (Berk.) Singer 
Ch. cf. columellifer 

Ch. craterellus (Durieu & Lév.) Singer 

Ch. fragilis (Pat.) Singer 

Ch. galeatus (Berk. & M.A. Curtis) Singer 
Ch. liliputianus (Mont.) Singer 

Ch. magnus Halling 

Crinipellis actinophora (Berk. & Broome) Singer 
Cr. brasiliensis Arruda et al. 

Cr. brunneipurpurea Corner 

Cr. brunnescens Kerekes & Desjardin 

Cr. campanella (Peck) Singer 

Cr. cupreostipes Kerekes et al. 

Cr. dipterocarpi Singer 

Cr. furcata Kerekes et al. 

Cr. cf. iopus Singer 

Cr. malesiana Kerekes et al. 

Cr. maxima A.H. Sm. & M.B. Walters 

Cr. piceae Singer 

Cr. procera G. Stev. 

Cr. scabella (Alb. & Schwein.) Murrill 

Cr. setipes (Peck) Singer 

Cr. stipitaria (Fr.) Pat. 

Crinipellis sp. 

Crinipellis sp. 

Cr. tabtim Kerekes et al. 

Cr. trichialis (Lév.) Pat. 

Cr. zonata (Peck) Sacc. 

Marasmius apatelius Singer 

M. leucorotalis Singer 

M. rotula (Scop.) Fr. 

Moniliophthora aurantiaca 

M. canescens (Har. Takah.) Kerekes & Desjardin 
M. perniciosa (Stahel) Aime & Phillips-Mora 
M. roreri (Cif) H. C. Evans et al. 
Moniliophthora sp. 

Tetrapyrgos nigripes (Fr.) Horak 

T. subcinerea (Berk. & Broome) Horak 

T. subdendrophora (Redhead) Horak 


ORIGIN 
Malaysia 
Ecuador 
Italy 
Thailand 
Thailand 
Puerto Rico 
Colombia 
Malaysia 
Brazil 
Indonesia 
Indonesia 
Thailand 
Thailand 
Indonesia 
Papua NG 
Malaysia 

N. America 
New Zealand 


Europe 


Thailand/USA 


Germany 
Thailand 
Guyana 
Thailand 
Indonesia 
Canada 
Thailand 
Malaysia 
N. America 
Samoa 
Malaysia 
Peru 

Costa Rica 
N. America 
N. America 
S. E. Asia 


N. America 


ITS 
FJ167665 
AY916686 
FJ167664 
FJ 167662 
FJ167663 
AY916682 
FJ167666 
FJ167626 
AY317137 
FJ167646 
FJ 167627 
FJ167641 
FJ167651 
FJ167658 
FJ167639 
FJ167629 
FJ167633 
FJ167660 
FJ167635 
FJ167634 
AY571033 
AY916698 
AY916701 
FJ167643 
FJ167609 
AY916692 
EU935561 
FJ431253 
DQ182506 
JN692482 
FJ 167668 
AF335590 
AY916746 
AY916754 
DQ449942 
EF175552 
EF175521 


* ITS sequence from a German collection, nLSU from an Estonian collection 
**ITS sequence originated from a Thai collection, nLSU sequence from a North American collection. 


Moniliophthora aurantiaca sp. nov. (Samoa) ... 495 


nLSU 
FJ167665 


AY916680 
AF042647 
AF042630 


AM946420* 
AY916689** 
AY207194 


AY916690 


DQ457686 
JN692483 


AY916744 


AF261337 


496 ... Kropp & Albee-Scott 


2003). The potential scale reduction factors for all convergence statistics approached 
1.001 for all parameters. Posterior probability support measures <50% are not shown. 
Tetrapyrgos was used to root the consensus tree. 


Results 

The results of the Bayesian analysis show that the undescribed species 
clusters with the Moniliophthora clade, which has very good posterior 
probability support (Fic. 1). Certain other taxa, such as Crinipellis aff. iopus and 
Crinipellis brasiliensis (which have not yet been transferred to Moniliophthora), 
also clustered within this clade. Our analysis indicates that M. aurantiaca 
has a stronger affinity with the neotropical members of the clade such as 
M. roreri than it does with the Australasian representatives such as M. 
canescens or Crinipellis aff. iopus. Moniliophthora aurantiaca is closely related to 
M. roreri, which has a neotropical distribution. In addition, two other neotropical 
taxa, M. perniciosa and Crinipellis brasiliensis, appear to be close relatives of 
M. aurantiaca based on our analyses. 

The species of Chaetocalathus and Marasmius formed two well-supported 
clades basal to Crinipellis and Moniliophthora. In our analysis, the members of 
Moniliophthora occupy a clade derived from within Crinipellis. Based on these 
results, accepting Moniliophthora as a genus renders Crinipellis paraphyletic, 
although the node separating Crinipellis clade 1 from Crinipellis clade 2 and 
Moniliophthora is not well supported (Fie. 1). 


Discussion 

Moniliophthora aurantiaca is an unusually conspicuous element of the 
mycobiota of American Samoa. It stands out primarily because of its bright 
orange colors but also because it fruits commonly in littoral forests. ‘Thus far, it 
has been found only in a narrow strip of damp littoral forest situated along the 
shoreline on the islands of Tutuila and Ta’u, American Samoa. In one instance, 
it was found among trees growing on a rocky outcrop a few meters above the 
water but still at the shoreline. That M. aurantiaca was never found outside 
the littoral zone in spite of an abundance of apparently suitable moist woody 
substrates in nearby higher elevation forests suggests a halophilic or maritime 
beach ecology. Known thus far only from American Samoa, M. aurantiaca may 
be endemic to the Samoan Islands, but its distribution is still poorly known and 
further work is needed to confirm whether it is really an endemic species. 

The species belongs to the Moniliophthora/Crinipellis complex that currently 
comprises five Moniliophthora species and 163 Crinipellis species (http.//www. 
indexfungorum.org/). Of these, M. aurantiaca macroscopically most closely 
resembles the South American species Crinipellis ticoi Halling, which is also 
orange in color. Despite their macroscopic similarities, C. ticoi differs from 
M. aurantiaca microscopically in larger spores (12.1-14.3 x 5-7.1 um) and 


Moniliophthora aurantiaca sp. nov. (Samoa) ... 497 


Tetrapyrgos nigripes 
Tetrapyrgos subcinerea 
Tetrapyrgos subdendrophora 
Marasmius apatelius 
Marasmius leucorotalis 
0.78 L. Marasmuus rotula 
0.94 Chaetocalathus cf columellifer 
Chaetocalathus magnus 
Chaetocalathus columellifer 
1.00 7 Chaetocalathus craterellus 
A068 Chaetocalathus galeatus 
0.97 | - Chaetocalathus fragilis 
1.00 | Chaetocalathus liliputianus 
Crinipellis zonata (Canada) 
0.99 | 1.00 Crinipellis procera (New Zealand) 
sche Crinipellis furcata (Indonesia) 
Crinipellis campanella 
Crinipellis maxima 
Moniliophthora sp (North America) 


snyjepesojovyD 


T sijedruna 


0.63 Crinipellis aff iopus (Papua New Guinea) = 
oe Moniliophthora canescens (Malaysia) = 

ae Moniliophthora aurantiaca (Samoa) ep 

1.00 ti Moniliophthora roreri (Costa Rica) a 

1.00 Moniliophthora perniciosa (Central America) S 

0.80 1.00 Crinipellis brasiliensis (Brazil) 4 


1.00 + Crinipellis malesiana (Malaysia) 
o99| | Crinipellis sp (Thailand) 
Crinipellis trichialis (Indonesia) 
100] 1 Crinipellis scabella (Europe) 
1.00 § Crinipellis stipitaria (Germany) 
1.00 |, oor Crinipellis piceae (North America) 
Crinipellis setipes (Thailand) 
Crinipellis brunneipurpurea (Indonesia) 
098 |_| 1.00 r Crinipellis sp (Guyana) 
Crinipellis actinophora (Malaysia) 
Crinipellis brunnescens (Indonesia) 
0.65 Crinipellis cupreostipes (Thailand) 
Crinipellis dipterocarpi (Thailand) 
1.00 L_ Crinipellis tabtim (Thailand) 


Z siffediursD 


1.00 


0.4 1.00 


FiGuRE 1. Phylogram from a Bayesian analysis of a combined ITS and nLSU dataset for Crinipellis, 
Moniliophthora, and Chaetocalathus. Support measures are shown for nodes with 60 percent or 
greater posterior probability support. Members of Tetrapyrgos were used to root the phylogram. 


cheilocystidia that lack the apical appendages present on the cheilocystidia 
of M. aurantiaca (Fic. 3). One other orange Crinipellis is the African 
C. hygrocyboides (Henn.) Singer. Neither Hennings (1902) nor Singer (1989) 
gives microscopic details or a detailed description for this fungus, making 
comparisons with other taxa difficult. Since Halling (1993) reports that spores 
and cystidia were not observed in the type material of C. hygrocyboides from 
the Muséum National d’Histoire Naturelle, we have studied isotype material of 
C. hygrocyboides (=Marasmius hygrocyboides Henn.) from the Royal Botanical 
Gardens, Kew. The specimen resembles M. aurantiaca but differs by having a 
mixture of long dextrinoid hairs and clavate, thin-walled dextrinoid cystidia 
on the stipe surface and similar, but slightly smaller, spores. We were unable to 


498 ... Kropp & Albee-Scott 


reliably study cheilocystidia in the C. hygrocyboides material due to difficulty 
reviving the material. However, all cells at the lamellar edges appeared to 
be clavate and different from the cheilocystidia with apical appendages of 
M. aurantiaca. It would be very useful to have molecular data for this species 
to determine whether it belongs to the Moniliophthora clade. Another similar 
taxon is M. canescens, which has orange hues when wet but is typically brownish; 
additional differences include an initially white stipe that becomes brown and 
narrower spores (Takahashi 2000). Crinipellis malesiana, which also develops 
orange colors in age (Kerekes & Desjardin 2009), is phylogenetically separate 
from M. aurantiaca (Fic. 1). 

Based on our phylogenetic analysis, M. aurantiaca falls within the 
Moniliophthora clade of Aime & Phillips-Mora (2005). These workers 
found that fungi belonging to Moniliophthora, which had been used for an 
anamorphic pathogen of cocoa (Evans et al. 1978), are not only closely allied to 
certain members of Crinipellis but form a distinct lineage with Crinipellis in the 
Marasmiaceae for which they proposed using Moniliophthora as a generic name. 
The genus currently includes both conidial and agaricoid taxa. The agaricoid 
members of Moniliophthora are usually relatively brightly colored species that 
would otherwise have been placed in section lopodinae of Crinipellis. 

In contrast, our analysis indicates that Moniliophthora does not occupy a 
distinct lineage but is derived from within Crinipellis. In this case, accepting 
Moniliophthora as a genus makes Crinipellis paraphyletic (Fic. 1). Based on 
our results, one could abandon Moniliophthora and transfer everything in it, 
including the anamorphic species, to Crinipellis. However, one cannot ignore 
that the ITS analysis of Kerekes & Desjardin (2009) and the 5-gene phylogeny 
of Aime & Phillips-Mora (2005) both agree that the Moniliophthora clade forms 
a distinct lineage. In addition, the node separating the two Crinipellis clades 
shown in our phylogram has weak posterior probability support (Fic. 1). Given 
the discrepancy between our analysis and the other two studies, further work 
is needed to clarify relationships in the Moniliophthora/Crinipellis complex. 
For now, the preponderant information indicates that the Moniliophthora clade 
forms a distinct lineage and until further work is done we retain Moniliophthora 
as a genus in the sense of Aime & Phillips-Mora (2005). 

Caution must be used when using our phylogram to identify biogeographical 
patterns. Nonetheless, the close affinity of M. aurantiaca to the pathogenic South 
and Central American members of the Moniliophthora clade is well supported 
and implies that either M. aurantiaca or an ancestral taxon originated in the 
neotropics (Fic. 1). This is somewhat unexpected, because we earlier found 
that another Samoan fungus, Inocybe tauensis Kropp & Albee-Scott (2010), has 
paleotropical roots and because Samoan plant communities have Australasian 
ties (Van Balgooy et al. 1996). 


Moniliophthora aurantiaca sp. nov. (Samoa) ... 499 


Regardless of where M. aurantiaca originated, long-distance dispersal was 
clearly involved because the islands are relatively young and have never been 
connected to a land mass (McDougall 1985). In recent times, humans could have 
carried M. aurantiaca to the Samoan Islands on plants or in woody materials 
from almost any part of the world. However, long-distance dispersal might also 
have occurred via other well-known mechanisms. Rafting is one potential means 
of transport because M. aurantiaca grows in woody debris and could be carried 
long distances by floating wood. Rafting is known to transport organisms and is 
thought to have resulted in the dispersal and subsequent cladogenesis of snails 
across large areas of the Pacific Ocean, including an endemic Samoan snail 
(Donald et al. 2005). In another instance, an oyster species is thought to have 
been carried via rafting from Chile to New Zealand (Foighil et al. 1999). Thus, 
a plausible mechanism exists for the dispersal of fungi from the Neotropics to 
Samoa. The mycelium of M. aurantiaca could have been carried within woody 
material from South America to Samoa via the South Equatorial Current that 
has a general westward flow across tropical portions of the South Pacific. Air- 
borne spores are another means by which agarics could potentially cross the 
Pacific and it is clear that fungal spores can travel long distances with air masses 
(Brown & Hovmeller 2002). If M. aurantiaca has neotropical roots, then its 
spores or those of an ancestral taxon would have needed to cross the Pacific 
from the east to get to the Samoan Islands. ‘The easterly trade winds of the 
equatorial South Pacific provide another plausible mechanism by which this 
could have happened. 


Taxonomy 


Moniliophthora aurantiaca Kropp & Albee-Scott, sp. nov. Fics 2, 3 
MyYcoBANK MB563345 


Differs from Crinipellis ticoi by its smaller basidiospores and cheilocystidia with apical 
appendages and from Crinipellis hygrocyboides by its short, moderately thick-walled 
setae-like stipe hairs and the absence of clavate caulocystidia. 


Type: American Samoa, National Park of American Samoa, Tau Unit, in littoral forest 
along trail to Siu point, on woody debris, 17 May 2009, leg. B.R. Kropp BK17-May- 
2009-20a (Holotype UTC253631; GenBank JN692482, JN692483). 


ETYMOLOGy: aurantiaca refers to the bright orange coloration of the pileus. 


PiLEus 3-15 mm diameter, convex with margin somewhat inrolled at first then 
broadly convex to nearly plane, disc with a small umbo, sometimes slightly 
depressed around the umbo, translucent-striate or slightly sulcate from the 
margin nearly to the disk, surface matted fibrillose; bright red orange (78A, 
2.5YR 7/12) at the center, becoming pale orange (54A, 5YR 8/8) toward 
the margin especially in age; when dry, pale orange with the umbo darker. 
LAMELLAE narrowly attached, subdistant, pale orange (65A, 5YR 8/8), very 


500 ... Kropp & Albee-Scott 


FiGuRE 2. Moniliophthora aurantiaca. Basidiomata: A. UTC253824; B. UTC253809. Scale = 1 cm. 


pale orange when dry; lamellulae in 1-2 series. Stipe 5-8 x 0.25-1 mm, 
central, terete, equal, densely pruinose, concolorous with the lamellae except 
for the base which is often brown, extreme base on some specimens strigose, 
stipe inserted for most specimens. PILEIPELLIs a cutis of fairly loosely woven 
light brown, lightly encrusted hyphae 5-8 um wide, not well differentiated 


Moniliophthora aurantiaca sp. nov. (Samoa) ... 501 


FiGuRE 3. Moniliophthora aurantiaca. A. Basidiospores B. Basidia C. Stipe hairs D. Pileipellis hairs 
E. Basidioles F. Cheilocystidia. Scales: D = 50 um; A-C, E, F = 10 um. 


from the pileus context; numerous long, dextrinoid hairs present, these with 
a basal clamp and 400-600 x 4-5 um, mostly hyaline not greenish in KOH; 
context not dextrinoid. LAMELLAR TRAMA subparallel to interwoven; hyphae 
hyaline sometimes lightly encrusted, clamped, 4-8 um wide; context not 
dextrinoid. STIPE TISsUE parallel, smooth, hyaline, hyphae 4-8 um wide; 
dextrinoid to weakly dextrinoid; stipe hairs numerous, covering the stipe apex 
and base, dextrinoid to weakly dextrinoid, short and moderately thick-walled, 
resembling setae, 52-85 x 5-10 um. BASIDIOSPORES 7.5-(8.7)-11.0 x 4-(4.6)- 
6.0 um, Q = 1.8-2.1, amygdaliform, thin-walled, smooth, hyaline, inamyloid. 
BASIDIA 26-30 x 6-7 um, clavate, typically four-spored. BASIDIOLES 22-25 x 
4-5 um, clavate. PLEUROCYSTIDIA none. CHEILOCYSTIDIA 20-22 x 5-8 um, 
numerous, hyaline, thin-walled, typically with several irregular apical, finger- 
like appendages. CLamps present. RHIZOMORPHS absent. 
HABITAT AND DISTRIBUTION occurring on fallen twigs and other woody 

material in littoral forest. 

ADDITIONAL SPECIMENS EXAMINED—AMERICAN SAMOA. TuTUILA, near Vaitogi, 

on woody debris in wooded area near shoreline, 11 May 2009, leg. B.R. Kropp, BK11- 


May-2009-11 (UTC253809); National Park of American Samoa, Tutuila Unit, on woody 
debris in littoral forest along trail to Pola Island, 12 May 2009, leg. B.R. Kropp, BK12- 


502 ... Kropp & Albee-Scott 


May-2009-12 (UTC253824); BK12-May-2009-16 (UTC253828); BK12-May-2009-18 
(UTC253830); BK12-May-2009-17 (UTC253829). Ta’u, National Park of American 
Samoa, Tau Unit, on woody debris in littoral forest along trail to Siu point, 15 May 
2009, leg. B.R. Kropp, BK15-May-2009-10a (UTC255959). 


Acknowledgements 

The National Park of American Samoa and the Department of Marine and Wildlife 
Resources in American Samoa are thanked for facilitating the necessary collecting 
permits and for providing helpful advice about collecting within the national park. Dr. 
M.C. Aime and Dr. T.J. Baroni are gratefully acknowledged for their peer reviews of this 
article. 


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(chocolate, Theobroma cacao) form a new lineage of Marasmiaceae. Mycologia 97: 1012-1022. 
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Brooks E. 2004. Wood decay fungi: pests and diseases of American Samoa. Land Grant Technical 
Report No. 11, American Samoa Community College. 2 p. 

Brooks FE. 2006. List of Plant Diseases in American Samoa. Land Grant Technical Report No. 44, 
American Samoa Community College. 59 p. 

Brown JKM, Hovmeller MS. 2002. Aerial dispersal of pathogens on the global and continental 
scales and its impact on plant disease. Science 297: 537-541. 
http://doi.dx.org/10.1126/science.1072678 

Donald KM, Kennedy M, Spencer HG. 2005. Cladogenesis as the result of long-distance 
rafting events in South Pacific topshells (Gastropoda, Trochidae). Evolution 59: 1701-1711. 
http://doi.dx.org/10.1554/04-553.1 

Evans HC, Stalpers JA, Samson RA, Benny GL. 1978. On the taxonomy of Monilia roreri, an 
important pathogen of Theobroma cacao in South America. Canadian Journal of Botany 56: 
2528-2532. http://doi.dx.org/10.1139/b78-305 

Foighil D, Marshall BA, Hilbish TJ, Pino M. 1999. Trans-Pacific range extension by rafting 
is inferred for the Flat Oyster Ostrea chilensis. The Biological Bulletin 196: 122-126. 
http://doi.dx.org/10.2307/1542557 

Halling R. 1993. Two new Crinipellinae (Tricholomataceae: Marasmiae) from South America. 
Mycotaxon 47: 379-385. 

Hennings P. 1902. Fungi camerunensis novi. III. Botanische Jahrbiicher fiir Systematik 30: 39-57. 

Kerekes JF, Desjardin DE. 2009. A monograph of the genera Crinipellis and Moniliophthora from 
Southeast Asia including a molecular phylogeny of the nrITS region. Fungal Diversity 37: 
101-152. 

Kropp BR, Albee-Scott S. 2010. Inocybe tauensis, a new species from the Samoan Archipelago 
with biogeographic evidence for a Paleotropical origin. Fungal Biology 114: 790-796. 
http://doi.dx.org/10.1016/j.funbio.2010.07.005 

Kropp BR, Hansen D, Flint KM, Thomson SV. 1996. Artificial inoculation and colonization of 
Dyer’s Woad (Isatis tinctoria) by the systemic fungus Puccinia thlaspeos. Phytopathology 86: 
891-896. http://doi.dx.org/10.1094/Phyto-86-891 

McDougall I. 1985. Age and evolution of the volcanoes of Tutuila, AS. Pacific Science 39: 
311-320. 


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Maddison DR. 1994. Phylogenetic methods for inferring the evolutionary history and processes 
of change in discretely valued characters. Annual Review of Entomology 39: 267-292. 
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Moncalvo J-M, Lutzoni FM, Rehner SA, Johnson J, Vilgalys R. 2000. Phylogenetic relationships of 
agaric fungi based on nuclear large subunit ribosomal DNA sequences. Systematic Biology. 49: 
278-305. http://doi.dx.org/10.1093/sysbio/49.2.278 

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Ronquist F, Huelsenbeck JP. 2003. MrBayes 3: Bayesian phylogenetic inference under mixed 
models. Bioinformatics 19: 1572-1574. http://doi.dx.org/10.1093/bioinformatics/btg180 

Singer R. 1989. New taxa and new combinations of Agaricales (Diagnoses fungorum novorum 
Agaricalium IV). Fieldiana, Botany New Series 21: 1-133. 
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Takahashi H. 2000. Three new species of Crinipellis found in Iriomote Island, southwestern Japan, 
and central Honshu, Japan. Mycoscience 41: 171-182. http://doi.dx.org/10.1007/BF02464328 

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and historical distribution patterns in plants. 191-213, in: A Keast, SE Miller (eds). The origin 
and evolution of Pacific island biotas, New Guinea to Eastern Polynesia: patterns and processes. 
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Wanscher JH, Kornerup A. 1991. Farver i farver: bestemmelse af farver. Politikens Forlag, 
Copenhagen. 248 p. 

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methods and applications. Academic Press, Inc., New York. 


ISSN (print) 0093-4666 © 2012 Mycotaxon, Ltd. ISSN (online) 2154-8889 


MYCOTAXON 


http://dx.doi.org/10.5248/119.505 
Volume 120, pp. 505-506 April-June 2012 


Regional annotated mycobiotas new to the Mycotaxon website 


ABASTRACT — Five new species distribution lists are added to the MycoTaxon ‘web- 
list’ page covering Coprinaceae & Strophariaceae and Bolbitiaceae & Crepidotaceae in 
Argentina (both by Niveiro & Albert6); macromycetes from Collestrada forest ecosystems 
in Perugia, Italy (by Angelina & al.); lichens from Pollino National Park, Italy (by 
Puntillo & al.); and saprobes on Alnus alnobetula in the Swiss alps (by Senn-Irlet & al.). 
This brings to 94 the free access mycobiotas now available on the MycoTaxon website 
<http://www.mycotaxon.com/resources/weblists.htm]>. 


SOUTH AMERICA 
Argentina 


Niveiro, N.; Alberté, E. Checklist of the Argentinean Agaricales 2. Coprinaceae and 
Strophariaceae. 38 p. 
Asstract — A checklist of species belonging to the families Coprinaceae and 
Strophariaceae was made for Argentina. The list includes all species published till year 
2011. Twenty-one genera and 251 species were recorded, 121 species from the family 
Coprinaceae and 130 from Strophariaceae. 


Niveiro N. & E. Alberté. Checklist of the Argentine Agaricales 3. Bolbitiaceae and 
Crepidotaceae. 28 p. 
Asstract — A checklist of Agaricales species belonging to Bolbitiaceae and 
Crepidotaceae has been made for Argentina. The list included all species published 
until 2011. Fourteen genera and 187 species are recorded, 94 species from the 
Bolbitiaceae and 93 from Crepidotaceae. 


EUROPE 
Italy 


Angelini, Paola, Giancarlo Bistocchi, Andrea Arcangeli & Roberto Venanzoni. 
Preliminary checklist of the macromycetes from Collestrada forest ecosystems 
in Perugia (Italy). 14 p. 
ABSTRACT — A preliminary taxonomic list of the macromycetes growing in forest 
ecosystems in Perugia (Italy) is presented based on mycological research carried 


506 ... New regional mycobiotas online 


out in the most widespread local plant communities from the forest of Collestrada: 
Quercus spp. woodlands, Carpinus betulus L. woodland and plantations with Pinus 
pinea e/o Pinus pinaster. In the period from Jan. 2011 to Dec. 2011 133 taxa belonging 
to 70 genera were recorded. For each taxa the following items were reported: Latin 
name, author, WGS-84 Global Position System (GPS) coordinates, coordinate grids 
from a Google Earth Collestrada image, date of the survey and habitat. This work 
contributes to the Umbrian regional checklist, which will eventually be integrated 
with the Italian national checklist. 


Puntillo, D., M. Puntillo, G. Potenza & S. Fascetti. New, rare, and noteworthy lichens 
in the Pollino National Park (Basilicata, southern Italy). 13 pp. 


Asstract — 42 lichens new to the Basilicata Region were found in the 
territory of the Pollino National Park (southern Italy). There are two 
very interesting groups of species: the first, the Caliciales, indicator of the 
ecological continuity of the forest, are present on old trees (Abies alba Mill.). 
The second one consists of foliicolous species that are present in a small 
humid valley with subtropical microclimatic conditions. From the results 
of this work, the number of taxa of the lichen flora of Basilicata rises to 558 
species, with an increase of 9%. 


Switzerland 


Senn-Irlet, Beatrice, Rolf Miirner, Elia Martini, Nicolas Kiiffer, Romano de Marchi & 
Guido Bieri. Saprobic fungi on wood and litter of Alnus alnobetula in the Swiss 
Alps. 34 pp. 


ABSTRACT — 246 species representing 73 genera and 90 species of ascomycetes, 
basidiomycetes being represented with 44 genera of aphyllophoralean fungi with 77 
species, 23 genera of agarics with 68 species and 8 genera of tremelloid fungi with 
12 species growing on wood and litter of Alnus alnobetula in Switzerland are given. 
Clitocybe and Mycena species dominate among the leaf litter inhabiting species. Fallen 
branches have the highest species diversity. The host-specific Peniophora aurantiaca is 
one of the most conspicuous and most frequent species. 


ISSN (print) 0093-4666 © 2012 Mycotaxon, Ltd. ISSN (online) 2154-8889 


MY COTAXON 


http://dx.doi.org/10.5248/120.507 
Volume 120, pp. 507-510 April-June 2012 


NOMENCLATURAL NOVELTIES AND TYPIFICATIONS 


PROPOSED IN MYCOTAXON 120 


Acrogenospora hainanensis Jian Ma & X.G. Zhang, p. 59 

Annulatascus menglensis D.M. Hu, L. Cai & K.D. Hyde, p. 82 

Aqualignicola vaginata D.M. Hu, L. Cai & K.D. Hyde, p. 84 

Arcyria galericulata B. Zhang & Yu Li, p. 402 

Basidiobolomycetes Humber, p. 484 

Bipolaris salkadehensis Anmadpour & Heidarian, p. 302 

Caeoma ahmadii Afshan, Niazi & Khalid, p. 241 

Caeoma khanspurense Khalid, Afshan & Niazi, p. 242 

Caeoma rosicola Afshan, Khalid & Niazi, p. 243 

Cantharellus zangii X.F. Tian, P.G. Liu & Buyck, p. 100 

Clavulicium hallenbergii Avneet P. Singh, J. Kaur & Dhingra, p. 353 

Colletotrichum populi C.M. Tian & Zheng Li, p. 283 

Conidiobolus sinensis Y. Nie, X.Y. Liu & B. Huang, p. 432 

Coriolopsis psila (Lloyd) Ryvarden, p. 228 

Crepidotus variabilis var. subsphaerosporus J.E. Lange 1940, 
lectotypified, epitypified, p. 425 

Entoloma aurantioquadratum C.K. Pradeep & K.B. Vrinda, p. 338 

Entoloma crassum C.K. Pradeep & K.B. Vrinda, p. 335 

Entoloma suaveolens C.K. Pradeep & K.B. Vrinda, p. 332 

Entomophthoromycetes Humber, p. 486 

Entomophthoromycota Humber, p. 481 

Erysiphe javanica Meeboon & S. Takam., p. 191 

Exobasidium ferrugineae Minnis, A.H. Kenn. & N.A. Goldberg, p. 455 

Fusarium bactridioides Wollenw. 1934, lectotypified, p. 418 

Glomus trufemii B.T. Goto, G.A. Silva & Oehl, p. 3 

Gomphus crassipes (L.M. Dufour) Maire 1937, lectotypified, p. 394 

Infundibulicybe lateritia (J. Favre) Vizzini & Contu, p. 368 

Infundichalara minuta Koukol, p. 346 

Inonotus multisetifer Abrahao & Gugliotta, p. 36 


508 ... MycoOTAXON 120 


Lactifluus subg. Piperati Verbeken, p. 449 

Lactifluus sect. Allardii (Hesler & A.H. Sm.) De Crop, p. 449 
Lactifluus sect. Aurantiifolii (Verbeken) Verbeken, p. 450 

Lactifluus sect. Phlebonemi (R. Heim ex Verbeken) Verbeken, p. 446 
Lactifluus sect. Piperati (Fr.) Verbeken, p. 449 

Lactifluus sect. Polysphaerophori (Singer) Verbeken, p. 445 

Lactifluus sect. Pseudogymnocarpi (Verbeken) Verbeken, p. 447 
Lactifluus sect. Rubroviolascentini (Singer) Verbeken, p. 447 
Lactifluus sect. Tomentosi (McNabb) Verbeken, p. 448 

Lactifluus acicularis (Van de Putte & Verbeken) Van de Putte, p. 444 
Lactifluus albocinctus (Verbeken) Verbeken, p. 445 

Lactifluus allardii (Coker) De Crop, p. 450 

Lactifluus angustus (R. Heim & Gooss.-Font.) Verbeken, p. 446 
Lactifluus arsenei (R. Heim) Verbeken, p. 446 

Lactifluus aurantiifolius (Verbeken) Verbeken, p. 450 

Lactifluus austrovolemus (Hongo) Verbeken, p. 444 

Lactifluus brunneoviolascens (Bon) Verbeken, p. 446 

Lactifluus brunnescens (Verbeken) Verbeken, p. 445 

Lactifluus caperatus (R. Heim & Gooss.-Font.) Verbeken, p. 450 
Lactifluus caribaeus (Pegler) Verbeken, p. 446 

Lactifluus carmineus (Verbeken & Walleyn) Verbeken, p. 447 
Lactifluus clarkeae (Cleland) Verbeken, p. 448 

Lactifluus cocosmus (Van de Putte & De Kesel) Van de Putte, p. 450 
Lactifluus crocatus (Van de Putte & Verbeken) Van de Putte, p. 444 
Lactifluus denigricans (Verbeken & Karhula) Verbeken, p. 447 
Lactifluus distantifolius (Van de Putte, Stubbe & Verbeken) Van de Putte, p. 444 
Lactifluus dwaliensis (K. Das, J.R. Sharma & Verbeken) K. Das, p. 449 
Lactifluus flammans (Verbeken) Verbeken, p. 445 

Lactifluus foetens (Verbeken & Van Rooij) Verbeken, p. 445 
Lactifluus glaucescens (Crossl.) Verbeken, p. 449 

Lactifluus goossensiae (Beeli) Verbeken, p. 445 

Lactifluus gymnocarpoides (Verbeken) Verbeken, p. 447 

Lactifluus gymnocarpus (R. Heim ex Singer) Verbeken, p. 445 
Lactifluus kivuensis (Verbeken) Verbeken, p. 448 

Lactifluus lamprocystidiatus (Verbeken & E. Horak) Verbeken, p. 444 
Lactifluus leucophaeus (Verbeken & E. Horak) Verbeken, p. 449 
Lactifluus longipilus (Van de Putte, H.T. Le & Verbeken) Van de Putte, p. 444 
Lactifluus longisporus (Verbeken) Verbeken, p. 447 

Lactifluus longivelutinus (X.H. Wang & Verbeken) X.H. Wang, p. 446 


NOMENCLATURAL NOVELTIES & TYPIFICATIONS ... 


Lactifluus luteolus (Peck) Verbeken, p. 446 

Lactifluus luteopus (Verbeken) Verbeken, p. 447 

Lactifluus medusae (Verbeken) Verbeken, p. 447 

Lactifluus nonpiscis (Verbeken) Verbeken, p. 446 

Lactifluus novoguineensis (Henn.) Verbeken, p. 449 

Lactifluus olivescens (Verbeken & E. Horak) Verbeken, p. 449 
Lactifluus paleus (Verbeken & E. Horak) Verbeken, p. 449 

Lactifluus pallidilamellatus (Montoya & Bandala) Van de Putte, p. 444 
Lactifluus phlebonemus (R. Heim & Gooss.-Font.) Verbeken, p. 446 
Lactifluus pinguis (Van de Putte & Verbeken) Van de Putte, p. 445 
Lactifluus pisciodorus (R. Heim) Verbeken, p. 446 

Lactifluus pseudogymnocarpus (Verbeken) Verbeken, p. 447 
Lactifluus pseudoluteopus (X.H. Wang & Verbeken) X.H. Wang, p. 448 
Lactifluus pseudovolemus (R. Heim) Verbeken, p. 448 

Lactifluus pumilus (Verbeken) Verbeken, p. 447 

Lactifluus putidus (Pegler) Verbeken, p. 446 

Lactifluus roseophyllus (R. Heim) De Crop, p. 449 

Lactifluus rubiginosus (Verbeken) Verbeken, p. 6 

Lactifluus rubrobrunnescens (Verbeken, E. Horak & Desjardin) Verbeken, p. 446 
Lactifluus rubroviolascens (R. Heim) Verbeken, p. 447 

Lactifluus rugatus (Kiihner & Romagn.) Verbeken, p. 448 

Lactifluus subclarkeae (Grgur.) Verbeken, p. 450 

Lactifluus subpiperatus (Hongo) Verbeken, p. 449 

Lactifluus tanzanicus (Karhula & Verbeken) Verbeken, p. 445 
Lactifluus tenuicystidiatus (X.H. Wang & Verbeken) X.H. Wang, p. 447 
Lactifluus veraecrucis (Singer) Verbeken, p. 445 

Lactifluus vitellinus (Van de Putte & Verbeken) Van de Putte, p. 445 
Lactifluus volemoides (Karhula) Verbeken, p. 448 

Lactifluus xerampelinus (Karhula & Verbeken) Verbeken, p. 448 
Leptogium subjuressianum Marcelli & Kitaura, p. 218 

Leptogium subjuressianum var. caparoense Kitaura & Marcelli, p. 220 
Manoharachariella indica Rajeshkumar & S.K. Singh, p. 44 
Moniliophthora aurantiaca Kropp & Albee-Scott, p. 499 

Nectria zangii Z.Q. Zeng & W.Y. Zhuang, p. 69 

Neozygitales Humber, p. 486 

Neozygitomycetes Humber, p. 485 

Octaviania violascens Choeyklin, Boonprat. & Somrith., p. 150 
Omphalina pyxidata var. cystidiata M. Curti, Contu & Vizzini, p. 364 
Paradoxa sinensis L. Fan & J.Z. Cao, p. 473 


509 


510... MYCOTAXON 120 


Paralepista abdita (Dé6rfelt) Vizzini, p. 262 

Paralepista ameliae (Arcang.) Vizzini, p. 262 

Paralepista biformis (Peck) Vizzini, p. 262 

Paralepista femoralis (H.E. Bigelow) Vizzini, p. 262 

Paralepista flaccida (Sowerby) Vizzini, p. 262 

Paralepista flaccida var. fibrillosa (Malencgon) Vizzini, p. 262 

Paralepista gilva (Pers.) Vizzini, p. 262 

Paralepista lentiginosa (Fr.) Vizzini, p. 262 

Paralepista maculosa (Sacc.) Vizzini, p. 262 

Paralepista pseudoparilis (Enderle & Contu) Vizzini, p. 263 

Paralepista shafferi (H.E. Bigelow) Vizzini, p. 263 

Paralepista splendens (Pers.) Vizzini, p. 263 

Paralepistopsis Vizzini, p. 257 

Paralepistopsis acromelalga (Ichimura) Vizzini, p. 261 

Paralepistopsis amoenolens (Malencon) Vizzini, p. 257 

Passalora aseptata R. Singh, Chaurasia, K. Shukla & Upadhyaya, p. 462 

Phaeocollybia nigripes Wartchow & V. Coimbra, p. 172 

Phlyctis communis Chitale & Makhija, p. 76 

Pisolithus capsulifer (Sowerby) Watling, Phosri & M.P. Martin 
(also epitypified, lectotypified), p. 202 

Pisolithus orientalis Watling, Phosri & M.P. Martin, p. 205 

Plectania seaveri M. Carbone, Agnello & LaGreca, p. 318 

Pluteus crenulatus Justo, Battistin & Angelini, p. 14 

Pluteus stenotrichus Justo, Battistin & Angelini, p. 17 

Postia subplacenta B.K. Cui, p. 232 


Pseudocercospora danaicola (Vienn.-Bourg.) Pirnia & Zare 
(also neotypified), p. 164 


Rossbeevera yunnanensis Orihara & M.E. Sm., p. 141 
Russula jilinensis G.J. Li & H.A. Wen, p. 51 
Septobasidium capparis S.Z. Chen & L. Guo, p. 269 
Septobasidium dacrydii S.Z. Chen & L. Guo, p. 274 
Septobasidium reevesiae S.Z. Chen & L. Guo, p. 272 
Sympodioplanus yunnanensis G.Z. Yang & Z.F. Yu, p. 289 
Terriera simplex Y.R. Lin, X.M. Gao & C.T. Zheng, p. 210 
Tuber microsphaerosporum L. Fan & Yu Li, p. 471 
Tubifera applanata Leontyev & Fefelov, p. 248 

Vararia longicystidiata Samita, Sanyal, Dhingra & Avneet P. Singh, p. 357 
Wrightoporia nigrolimitata Jia J. Chen, p. 296 


bad taxonomy 


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can KILL